Stabilized alpha helical peptides and uses thereof

ABSTRACT

Novel polypeptides and methods of making and using the same are described herein. The polypeptides include cross-linking (“hydrocarbon stapling”) moieties to provide a tether between two amino acid moieties, which constrains the secondary structure of the polypeptide. The polypeptides described herein can be used to treat diseases characterized by excessive or inadequate cellular death.

CLAIM OF PRIORITY

This application is a continuation of and claims priority from U.S.application Ser. No. 13/252,751, filed Oct. 4, 2011, which is acontinuation of U.S. application Ser. No. 12/233,555, filed Sep. 18,2008, which is a continuation of U.S. application Ser. No. 12/182,673,filed Jul. 30, 2008, now U.S. Pat. No. 8,198,405, which is acontinuation of U.S. application Ser. No. 10/981,873, filed Nov. 5,2004, now U.S. Pat. No. 7,723,469, which claims the benefit of U.S.Provisional Application Ser. No. 60/517,848, filed on Nov. 5, 2003, andU.S. Provisional Application Ser. No. 60/591,548, filed on Jul. 27,2004. These contents of these prior applications are hereby incorporatedby reference in their entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 30, 2014, isnamed 35224-705.304-Seqlist.txt and is 77 Kilo bytes in size.

BACKGROUND

Apoptosis, or programmed cell death, plays a critical role in thedevelopment and maintenance of homeostasis in all multicellularorganisms. Susceptibility to apoptosis varies markedly among cells andis influenced by both external and internal cellular events. Positiveand negative regulator proteins that mediate cell fate have beendefined, and dysregulation of these protein signaling networks has beendocumented in the pathogenesis of a wide spectrum of human diseases,including a variety of cancers. BCL-2 is the founding member of thisfamily of apoptotic proteins and was first identified at the chromosomalbreakpoint of t(14;18)(q32;q21) lymphomas (Bakhashi et al. 1985 Cell41:899; Cleary et al. 1985 Proc. Nat'l. Acad. Sci. USA 82:7439).

Gene rearrangement places BCL-2 under the transcriptional control of theimmunoglobulin heavy chain locus, generating inappropriately high levelsof BCL-2 and resultant pathologic cell survival. Such aberrations inapoptosis have been identified in lymphocytic and myelogenous leukemiasand a host of other malignancies, and have been linked to tumorprogression and acquired resistance to chemotherapy-induced apoptosis.The BCL-2 family of proteins has expanded significantly and includesboth pro- and anti-apoptotic molecules that provide the checks andbalances that govern susceptibility to cell death (FIG. 1).

Not surprisingly, apoptotic proteins have become key targets for thedevelopment of therapeutics to both prevent precipitous cell death indiseases of cell loss and activate cell death pathways in malignancy.

The BCL-2 family is defined by the presence of up to four conserved“BCL-2 homology” (BH) domains designated BH1, BH2, BH3, and BH4, all ofwhich include α-helical segments (Chittenden et al. 1995 EMBO 14:5589;Wang et al. 1996 Genes Dev. 10:2859). Anti-apoptotic proteins, such asBCL-2 and BCL-X_(L), display sequence conservation in all BH domains.Pro-apoptotic proteins are divided into “multidomain” members (e.g. BAK,BAX), which possess homology in the BH1, BH2, and BH3 domains, and the“BH3-domain only” members (e.g. BID, BAD, BIM, BIK, NOXA, PUMA), thatcontain sequence homology exclusively in the BH3 amphipathic α-helicalsegment. BCL-2 family members have the capacity to form homo- andheterodimers, suggesting that competitive binding and the ratio betweenpro- and anti-apoptotic protein levels dictates susceptibility to deathstimuli. Anti-apoptotic proteins function to protect cells frompro-apoptotic excess, i.e., excessive programmed cell death. Additional“security” measures include regulating transcription of pro-apoptoticproteins and maintaining them as inactive conformers, requiring eitherproteolytic activation, dephosphorylation, or ligand-inducedconformational change to activate pro-death functions. In certain celltypes, death signals received at the plasma membrane trigger apoptosisvia a mitochondrial pathway (FIG. 2). The mitochondria can serve as agatekeeper of cell death by sequestering cytochrome c, a criticalcomponent of a cytosolic complex which activates caspase 9, leading tofatal downstream proteolytic events. Multidomain proteins such asBCL-2/BCL-X_(L) and BAK/BAX play dueling roles of guardian andexecutioner at the mitochondrial membrane, with their activities furtherregulated by upstream BH3-only members of the BCL-2 family. For example,BID is a member of the “BH3-domain only” subset of pro-apoptoticproteins, and transmits death signals received at the plasma membrane toeffector pro-apoptotic proteins at the mitochondrial membrane. BID hasthe unique capability of interacting with both pro- and anti-apoptoticproteins, and upon activation by caspase 8, triggers cytochrome crelease and mitochondrial apoptosis. Deletion and mutagenesis studiesdetermined that the amphipathic α-helical BH3 segment of pro-apoptoticfamily members functions as a death domain and thus represents acritical structural motif for interacting with multidomain apoptoticproteins. Structural studies have demonstrated that the BH3 helixinteracts with anti-apoptotic proteins by inserting into a hydrophobicgroove formed by the interface of BH1, 2 and 3 domains. Activated BIDcan be bound and sequested by anti-apoptotic proteins (e.g., BCL-2 andBCL-X_(L)) and can trigger activation of the pro-apoptotic proteins BAXand BAK, leading to cytochrome c release and a mitochondrial apoptosisprogram.

BAD is also a “BH3-domain only” pro-apoptotic family member whoseexpression likewise triggers the activation of BAX/BAK. In contrast toBID, however, BAD displays preferential binding to anti-apoptoticmembers, BCL-2 and BCL-X_(L). Whereas the BAD BH3 domain exhibits highaffinity binding to BCL-2, BAD BH3 peptide is unable to activatecytochrome c release from mitochondria in vitro, suggesting that BAD isnot a direct activator of BAX/BAK. Mitochondria that overexpress BCL-2are resistant to BID-induced cytochrome c release, but co-treatment withBAD can restore BID sensitivity. Induction of mitochondrial apoptosis byBAD appears to result from either: (1) displacement of BAX/BAKactivators, such as BID and BID-like proteins, from the BCL-2/BCL-X_(L)binding pocket, or (2) selective occupation of the BCL-2/BCL-X_(L)binding pocket by BAD to prevent sequestration of BID-like proteins byanti-apoptotic proteins. Thus, two classes of “BH3-domain only” proteinshave emerged, BID-like proteins that directly activate mitochondrialapoptosis, and BAD-like proteins, that have the capacity to sensitizemitochondria to BID-like pro-apoptotics by occupying the binding pocketsof multidomain anti-apoptotic proteins.

The objective of identifying or generating small molecules to probeapoptotic protein functions in vitro and specifically manipulateapoptotic pathways in vivo has been challenging. High throughputscreening has identified several molecules that inhibit the interactionof the BAK BH3 domain with BCL-X_(L) at micromolar affinities. Inaddition to the potential drawback of identifying low affinitycompounds, the technique is limited in its ability to generate panels ofcompounds tailored to the subtle binding specificities of individualmembers of protein families. Alternate approaches to manipulatingapoptosis pathways have derived from peptide engineering, a techniquethat uses non-specific peptide sequence to generate compounds withdesired three-dimensional structures. One application of this techniqueinvolved the generation of “pro-apoptotic” α-helices comprised ofnonspecific peptide sequence used to induce cell death by disruptingmitochondrial membranes.

The alpha-helix is one of the major structural components of proteinsand is often found at the interface of protein contacts, participatingin a wide variety of intermolecular biological recognition events.Theoretically, helical peptides, such as the BH3 helix, could be used toselectively interfere with or stabilize protein-protein interactions,and thereby manipulate physiologic processes. However, biologicallyactive helical motifs within proteins typically have little structurewhen taken out of the context of the full-length protein and placed insolution. Thus, the efficacy of peptide fragments of proteins as in vivoreagents has been compromised by loss of helical secondary structure,susceptibility to proteolytic degradation, and inability to penetrateintact cells. Whereas several approaches to covalent helix stabilizationhave been reported, most methodologies involve polar and/or labilecrosslinks (Phelan et al. 1997 J. Am. Chem. Soc. 119:455; Leuc et al.2003 Proc. Nat'l. Acad. Sci. USA 100:11273; Bracken et al., 1994 J. Am.Chem. Soc. 116:6432; Yan et al. 2004 Bioorg. Med. Chem. 14:1403).Subsequently, Verdine and colleagues developed an alternatemetathesis-based approach, which employed α,α-disubstituted non-naturalamino acids containing alkyl tethers (Schafineister et al., 2000 J. Am.Chem. Soc. 122:5891; Blackwell et al. 1994 Angew Chem. Int. Ed.37:3281).

SUMMARY

This invention is based, in part, on the discovery that stablycross-linking a polypeptide having at least two modified amino acids (aprocess termed “hydrocarbon stapling”) can help to conformationallybestow the native secondary structure of that polypeptide. For example,cross-linking a polypeptide predisposed to have an alpha-helicalsecondary structure can constrain the polypeptide to its nativealpha-helical conformation. The constrained secondary structure canincrease resistance of the polypeptide to proteolytic cleavage and alsoincrease hydrophobicity. Surprisingly, in some instances, thepolypeptides can penetrate the cell membrane (e.g., through anenergy-dependent transport mechanism, e.g., pinocytosis). Accordingly,the crosslinked polypeptides described herein can have improvedbiological activity relative to a corresponding uncrosslinkedpolypeptide. For example the cross-linked polypeptide can include analpha-helical domain of a BCL-2 family member polypeptide (e.g., BID-BH3domain), which can bind to BAK/BAX and/or BCL-2/BCL-X_(L) to promoteapoptosis in a subject. In some instances, the crosslinked polypeptidecan be used to inhibit apoptosis. The cross-linked polypeptidesdescribed herein can be used therapeutically, e.g., to treat cancer in asubject.

In one aspect, the invention features polypeptide of formula (I),

wherein;

each R₁ and R₂ are independently H or a C₁ to C₁₀ alkyl, alkenyl,alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, orheterocyclylalkyl;

R₃ is alkyl, alkenyl, alkynyl; [R₄—K—R₄]_(n); each of which issubstituted with 0-6 R₅;

R₄ is alkyl, alkenyl, or alkynyl;

R₅ is halo, alkyl, OR₆, N(R₆)₂, SR₆, SOR₆, SO₂R₆, CO₂R₆, R₆, afluorescent moiety, or a radioisotope;

K is O, S, SO, SO₂, CO, CO₂, CONR₆, or

R₆ is H, alkyl, or a therapeutic agent;

n is an integer from 1-4;

x is an integer from 2-10;

each y is independently an integer from 0-100;

z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); and

each Xaa is independently an amino acid.

In some instances, the polypeptide binds to a BCL-2 family protein. Thepolypeptide can bind to an anti-apoptotic protein. The polypeptide canbind to a pro-apoptotic protein. The polypeptide can bind and activateBAX or BAK. In some instances, the polypeptide binds to a BH1, BH2and/or BH3 domain.

In some instances, the polypeptide activates cell death, for example thepolypeptide can trigger cytochrome c release and activate mitochondrialcell death.

In other instances, the polypeptide can inhibit cell death.

In some instances, the polypeptide includes a BH3 domain.

In some instances, x is 2, 3, or 6.

In some instances, each y is independently an integer between 3 and 15.

In some instances each y is independently an integer between 1 and 15.

In some instances, R₁ and R₂ are each independently H or C₁-C₆ alkyl.

In some instances, R₁ and R₂ are each independently C₁-C₃ alkyl.

In some instances, at least one of R₁ and R₂ are methyl. For example R₁and R₂ are both methyl.

In some instances R₃ is alkyl (e.g., C₈ alkyl) and x is 3.

In some instances, R₃ is C₁₁ alkyl and x is 6.

In some instances, R₃ is alkenyl (e.g., C₈ alkenyl) and x is 3.

In some instances x is 6 and R₃ is C₁₁ alkenyl.

In some instances, R₃ is a straight chain alkyl, alkenyl, or alkynyl.

In some instances R₃ is —CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—.

In certain embodiments the two alpha, alpha disubstituted stereocentersare both in the R configuration or S configuration (e.g., i, i+4cross-link), or one stereocenter is R and the other is S (e.g., i, i+7cross-link). Thus, where formula I is depicted as

the C′ and C″ disubstituted stereocenters can both be in the Rconfiguration or they can both be in the S configuration, for examplewhen X is 3. When x is 6, the C′ disubstituted stereocenter is in the Rconfiguration and the C″ disubstituted stereocenter is in the Sconfiguration. The R₃ double bond may be in the E or Z stereochemicalconfiguration.

In some instances R₃ is [R₄—K—R₄]_(n); and R₄ is a straight chain alkyl,alkenyl, or alkynyl.

In some instances, the polypeptide includes an amino acid sequence whichis at least about 60% (70%, 80%, 85%, 90%, 95% or 98%) identical to theamino acid sequence of EDIIRNI*RHL*QVGDSN_(L)DRSIW (SEQ ID NO:99),wherein * is a tethered amino acid. For example, there can be 1, 2, 3,4, 5 or more amino acid changes, e.g., conservative changes.

The tether can include an alkyl, alkenyl, or alkynyl moiety (e.g., C₅,C₈ or C₁₁ alkyl or a C₅, C₈ or C₁₁ alkenyl, or C₅, C₈ or C₁₁ alkynyl).The tethered amino acid can be alpha disubstituted (e.g., C₁-C₃ ormethyl). In some instances, the polypeptide can include an amino acidsequence which is at least about 60% (70%, 80%, 85%, 90%, 95% or 98%)identical to the amino acid sequence of EDIIRNIARHLA*VGD*N_(L)DRSIW (SEQID NO:92), wherein * is a tethered amino acid. For example, there can be1, 2, 3, 4, 5 or more amino acid changes, e.g., conservative changes. Insome instances, the polypeptide is transported through the cell membrane(e.g., through an active transport or endocytotic mechanism or bypassive transport). In certain embodiments the polypeptide does notinclude a Cys or Met.

In some embodiments the polypeptide comprises at least 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, or more contiguousamino acids of a BCL-2 or BCL-2 like domain, e.g., a BH3 domain orBH3-like domain, e.g., a polypeptide depicted in any of FIGS. 5a, 5b,and 28a-28h . Each [Xaa]_(y) is a peptide that can independentlycomprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25 or more contiguous amino acids of a BCL-2 or BCL-2 like domain,e.g., a BH3 domain or BH3-like domain, e.g., a polypeptide depicted inany of FIGS. 5a, 5b, and 28a-28h . [Xaa]_(x) is a peptide that cancomprise 3 or 6 contiguous amino acids of acids of a BCL-2 or BCL-2 likedomain, e.g., a BH3 domain or BH3-like domain, e.g., a polypeptidedepicted in any of FIGS. 5a, 5b, and 28a -28 h.

The polypeptide can comprise 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, 50 contiguous amino acids of acids of aBCL-2 or BCL-2 like domain, e.g., a BH3 domain or BH3-like domain, e.g.,a polypeptide depicted in any of FIGS. 5a, 5b, and 28a-28h (SEQ IDNos:1-118) wherein two amino acids that are separated by three aminoacids (or six amino acids) are replaced by amino acid substitutes thatare linked via R₃. Thus, at least two amino acids can be replaced bytethered amino acids or tethered amino acid substitutes. Thus, whereformula I is depicted as

[Xaa]_(y)′ and [Xaa]_(y)″ can each comprise contiguous polypeptidesequences from the same or different BCL-2 or BCL-2 like domains.

The invention features cross-linked polypeptides comprising 10 (11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50or more) contiguous amino acids of a BCL-2 or BCL-2 like domain, e.g., aBH3 domain or BH3-like domain, e.g., a polypeptide depicted in any ofFIGS. 5a, 5b, and 28a-28h (SEQ ID Nos:1-118) wherein the alpha carbonsof two amino acids that are separated by three amino acids (or six aminoacids) are linked via R₃, one of the two alpha carbons is substituted byR₁ and the other is substituted by R₂ and each is linked via peptidebonds to additional amino acids.

In some embodiments the polypeptide has apoptotic activity.

In some instances, the polypeptide also includes a fluorescent moiety orradioisotope.

In some instances, the polypeptide includes 23 amino acids; R₁ and R₂are methyl; R₃ is C₈ alkyl, C₁₁ alkyl, C₈ alkenyl, C₁₁ alkenyl, C₈alkynyl, or C₁₁ alkynyl; and x is 2, 3, or 6.

In some instances, the polypeptide includes an affinity label, atargeting moiety, and/or a biotin moiety.

In some instances, the polypeptide is a polypeptide selected from thegroup consisting of the polypeptides depicted in and of FIGS. 28a-h and5a-b (SEQ ID NOS: 1-118). In another aspect, the invention features amethod of making a polypeptide of formula (III), including

-   -   providing a polypeptide of formula (II); and

-   -   formula (II)

treating the compound of formula (II) with a catalyst to promote a ringclosing metathesis, thereby providing a compound of formula (III)

-   -   wherein

each R₁ and R₂ are independently H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl; heteroarylalkyl; or heterocyclylalkyl;

each n is independently an integer from 1-15;

x is 2, 3, or 6

each y is independently an integer from 0-100;

z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); and

each Xaa is independently an amino acid;

In some instances, the polypeptide binds to a BCL-2 family memberprotein.

In some instances, the catalyst is a ruthenium catalyst.

In some instances, the method also includes providing a reducing oroxidizing agent subsequent to the ring closing metathesis.

In some instances, the reducing agent is H₂ or the oxidizing agent isosmium tetroxide

In some instances, the invention features a method of treating a subjectincluding administering to the subject any of the compounds describedherein. In some instances, the method also includes administering anadditional therapeutic agent.

In some instances, the invention features a method of treating cancer ina subject including administering to the subject any of the compoundsdescribed herein. In some instances, the method also includesadministering an additional therapeutic agent.

In some instances, the invention features a library of the compoundsdescribed herein.

In some instances, the invention features a method of identifying acandidate compound for the promotion of apoptosis, including;

-   -   providing mitochondria;    -   contacting the mitochondria with any of the compounds described        herein;    -   measuring cytochrome c release; and    -   comparing the cytochrome c release in the presence of the        compound to the cytochrome c release in the absence of the        compound, wherein an increase in cytochrome c release in the        presence of the compound of formula I identifies the compound as        a candidate compound for the promotion of apoptosis.

In some instances, the invention features a polypeptide of the formula(IV),

wherein;

each R₁ and R₂ are independently H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;

R₃ is alkyl, alkenyl, alkynyl; [R₄—K—R₄]_(n) or a naturally occurringamino acid side chain;

each of which is substituted with 0-6 R₅;

R₄ is alkyl, alkenyl, or alkynyl;

R₅ is halo, alkyl, OR₆, N(R₆)₂, SR₆, SOR₆, SO₂R₆, CO₂R₆, R₆, afluorescent moiety, or a radioisotope;

K is O, S, SO, SO₂, CO, CO₂, CONR₆, or

R₆ is H, alkyl, or a therapeutic agent;

R₇ is alkyl, alkenyl, alkynyl; [R₄—K—R₄]_(n) or an naturally occurringamino acid side chain; each of which is substituted with 0-6 R₅;

n is an integer from 1-4;

x is an integer from 2-10;

each y is independently an integer from 0-100;

z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); and

each Xaa is independently an amino acid;

In some instances, the invention features a polypeptide of formula (I)

wherein;

each R₁ and R₂ are independently H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;

R₃ is alkyl, alkenyl, alkynyl; [R₄—K—R₄]_(n); each of which issubstituted with 0-6 R₅;

R₄ is alkyl, alkynyl, or alkynyl;

R₅ is halo, alkyl, OR₆, N(R₆)₂, SR₆, SOR₆, SO₂R₆, CO₂R₆; R₆, afluorescent moiety, or a radioisotope;

K is O, S, SO, SO₂, CO, CO₂, CONR₆, or

R₆ is H, alkyl, or a therapeutic agent;

n is an integer from 1-4;

x is an integer from 2-10;

each y is independently an integer from 0-100;

Z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); and

each Xaa is independently an amino acid;

wherein the polypeptide has at least 5% alpha helicity in aqueoussolution as determined by circular dichroism.

In some instances, polypeptide has at least 15%, at least 35%, at least50%, at least 60%, at least 70%, at least 80% or at least 90% alphahelicity as determined by circular dichroism.

In some instances, the invention features a polypeptide of formula (I),

wherein;

each R₁ and R₂ are independently H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;

R₃ is alkyl, alkenyl, alkynyl; [R₄—K—R₄]_(n); each of which issubstituted with 0-6 R₅;

R₄ is alkyl, alkynyl, or alkynyl;

R₅ is halo, alkyl, OR₆, N(R₆)₂, SR₆, SOR₆, SO₂R₆, CO₂R₆, R₆, afluorescent moiety, or a radioisotope;

K is O, S, SO, SO₂, CO, CO₂, CONR₆, or

R₆ is H, alkyl, or a therapeutic agent;

n is an integer from 1-4;

x is an integer from 2-10;

each y is independently an integer from 0-100;

z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); and

each Xaa is independently an amino acid;

wherein the polypeptide has at least a 1.25-fold increase in alphahelicity as determined by circular dichroism compared to the polypeptideof formula (IV)

wherein R₁, R₂, Xaa, x, y, and z are all as defined for formula (I)above.

In some instances, the polypeptide has at least a 1.5-fold, at least1.75 fold, at least 2.0-fold, at least 2.5-fold, at least 3-fold, or atleast 4-fold increase in alpha helicity as determined by circulardichroism compared to the polypeptide of formula (IV).

In some instances, the invention features a method of identifying acandidate compound for the inhibition of apoptosis, including;

-   -   providing mitochondria;    -   contacting the mitochondria with a compound described herein;    -   measuring cytochrome c release; and    -   comparing the cytochrome c release in the presence the compound        described herein to the cytochrome c release in the absence of        the compound described herein, wherein a decrease in cytochrome        c release in the presence of the compound described herein        identifies the compound described herein as a candidate compound        for the inhibition of apoptosis.

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable”, as used herein, refers to compounds which possessstability sufficient to allow manufacture and which maintains theintegrity of the compound for a sufficient period of time to be usefulfor the purposes detailed herein (e.g., therapeutic administration to asubject or generation of reagents to study or discover a biologicalpathway either in vitro or in vivo).

The compounds of this invention may contain one or more asymmetriccenters and thus occur as racemates and racemic mixtures, singleenantiomers, individual diastereomers and diastereomeric mixtures. Allsuch isomeric forms of these compounds are expressly included in thepresent invention. The compounds of this invention may also berepresented in multiple tautomeric forms, in such instances, theinvention expressly includes all tautomeric forms of the compoundsdescribed herein (e.g., alkylation of a ring system may result inalkylation at multiple sites, the invention expressly includes all suchreaction products). All such isomeric forms of such compounds areexpressly included in the present invention. All crystal forms of thecompounds described herein are expressly included in the presentinvention.

The term “amino acid” refers to a molecule containing both an aminogroup and a carboxyl group. Suitable amino acids include, withoutlimitation, both the D- and L-isomers of the 20 common naturallyoccurring amino acids found in peptides (e.g., A, R, N, C, D, Q, E, G,H, I, L, K, M, F, P, S, T, W, Y, V (as known by the one letterabbreviations)) as well as the naturally occurring and unnaturallyoccurring amino acids prepared by organic synthesis or other metabolicroutes.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide (e.g., a BH3 domain)without abolishing or substantially altering its activity. An“essential” amino acid residue is a residue that, when altered from thewild-type sequence of the polypeptide, results in abolishing orsubstantially abolishing the polypeptide activity.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a BH3 polypeptide, forexample, is preferably replaced with another amino acid residue from thesame side chain family.

The symbol “

” when used as part of a molecular structure refers to a single bond ora trans or cis double bond.

The term “amino acid side chain” refers to a moiety attached to theα-carbon in an amino acids. For example, the amino acid side chain foralanine is methyl, the amino acid side chain for phenylalanine isphenylmethyl, the amino acid side chain for cysteine is thiomethyl, theamino acid side chain for aspartate is carboxymethyl, the amino acidside chain for tyrosine is 4-hydroxyphenylmethyl, etc. Othernon-naturally occurring amino acid side chains are also included, forexample, those that occur in nature (e.g., an amino acid metabolite) orthose that are made synthetically (e.g., an alpha di-substituted aminoacid).

The term polypeptide encompasses two or more naturally occurring orsynthetic amino acids linked by a covalent bond (e.g., a amide bond).Polypeptides as described herein include full length proteins (e.g.,fully processed proteins) as well as shorter amino acids sequences(e.g., fragments of naturally occurring proteins or syntheticpolypeptide fragments).

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine. The term “alkyl” refers to a hydrocarbon chain that may be astraight chain or branched chain, containing the indicated number ofcarbon atoms. For example, C₁-C₁₀ indicates that the group may have from1 to 10 (inclusive) carbon atoms in it. In the absence of any numericaldesignation, “alkyl” is a chain (straight or branched) having 1 to 20(inclusive) carbon atoms in it. The term “alkylene” refers to a divalentalkyl (i.e., —R—).

The term “alkenyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group may have from 2 to 10(inclusive) carbon atoms in it. The term “lower alkenyl” refers to aC₂-C₈ alkenyl chain. In the absence of any numerical designation,“alkenyl” is a chain (straight or branched) having 2 to 20 (inclusive)carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group may have from 2 to 10(inclusive) carbon atoms in it. The term “lower alkynyl” refers to aC₂-C₈ alkynyl chain. In the absence of any numerical designation,“alkynyl” is a chain (straight or branched) having 2 to 20 (inclusive)carbon atoms in it.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may besubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, whereinthe cycloalkyl group additionally may be optionally substituted.Preferred cycloalkyl groups include, without limitation, cyclopropyl,cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl,cycloheptyl, and cyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring may be substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like. The term “heteroarylalkyl” or the term“heteroaralkyl” refers to an alkyl substituted with a heteroaryl. Theterm “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring may be substituted by a substituent. Examples ofheterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, tetrahydrofuranyl, and the like.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, aryl, heterocyclyl, or heteroaryl group at any atom of thatgroup. Suitable substituents include, without limitation, halo, hydroxy,mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy,alkanesulfonyl, alkylcarbonyl, and cyano groups.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 depicts BCL-2 family members having one or more conserved BCL-2homology (BH) domains.

FIG. 2 depicts a model of BID-mediated mitochondrial apoptosis.TNF-RI/Fas induces cleavage of BID, which translocates to themitochondria and trigger apoptosis.

FIG. 3 depicts a synthetic strategy for the generation of chiralα,α-disubstituted non-natural amino acids containing olefinic sidechains.

FIG. 4a depicts chemical structures of certain non-natural amino acids.

FIG. 4b depicts the crosslinking of synthetic amino acids at positions iand i+4 and i and i+7 by olefin metathesis.

FIG. 5a depicts SAHB3 compounds generated by non-natural amino acidsubstitution and olefin metathesis (SEQ ID NOs 92-96, 118, 119, 97-108,120, 121, 110, 111, 122, and 123, respectively).

FIG. 5b depicts certain crosslinked peptides used in the studiesdescribed herein (SEQ ID NOs 112-117, respectively).

FIG. 6A depicts the results of a study showing circular dichroismspectra of BH3 domain peptides of selected BCL-2 family members. FIG. 6Bdepicts the degree of α-helicity of BH3 domain peptides of selectedBCL-2 family members.

FIG. 7A depicts the results of a study showing that chemicalcrosslinking enhances the alpha helicity of SAHB3BID compounds comparedto the unmodified BID BH3 peptide. FIG. 7B depicts the results of astudy showing the degree of alpha helicity of SAHB3BID compoundscompared to the unmodified BID BH3 peptide.

FIG. 8A depicts the results of a study showing that a gly→glu mutant ofSAHB3BIDA polypeptide displays similar helical contact to thecorresponding gly containing polypeptide. FIG. 8B depicts the degree ofalpha helicity of a gly→glu mutant of SAHB3BIDA polypeptide compared tothe corresponding gly containing polypeptide.

FIG. 9 depicts the results of a study showing that truncation of the23-mer SABH3_(BID)B (“SAHB3b”) to a 16-mer results in loss ofα-helicity.

FIG. 10a depicts the results of a study showing that the kinetics of invitro trypsin proteolysis is retarded 3.5-fold by the SABH3_(BID)Acrosslink.

FIG. 10b depicts the results of a study of ex vivo serum stability ofpeptides, demonstrating a 10-fold increase in half-life of thecross-linked peptide compared to the unmodified peptide.

FIG. 10c depicts the results of an in vivo study showing thatSAHB3_(BID)A is maintained at higher serum concentrations over timecompared to BID BH3 peptide.

FIG. 11a depicts the results of a study showing that SAHB3_(BID)peptides display high affinity binding to GST-BCL2 in a fluorescencepolarization competitive binding assay.

FIG. 11b depicts the results of a study showing that the negativecontrol Gly to Glu point mutants of SAHB3_(BID)A and B are relativelypoor binders.

FIG. 11c depicts the results of a study showing that truncation ofSAHB3_(BID)B from a 23-mer to a 16-mer results in a more than 6-folddrop in K_(i), coincident with a significant decrease in percenthelicity of the truncated compound.

FIG. 11d depicts the results of a BCL-2 fluorescence polarization directbinding assay demonstrating a more than 6-fold enhancement in bindingaffinity of SAHB3_(BID)A compared to unmodified BID BH3.

FIG. 11e depicts the results of a BAX fluorescence polarization directbinding assay demonstrating that incorporation of a crosslink results inmeasurable binding of SAHB3_(BID)A and SAHB3_(BID(G→E))A to amultidomain pro-apoptotic BCL-2 family member. The unmodified BID BH3peptide shows no binding.

FIG. 11f depicts HSQC spectra that demonstrate a conformational changein ¹⁵N-labeled BCL-X_(L) upon SAHB3_(BID)A binding, which is similar tothat seen upon BID BH3 binding, confirming that SAHB3_(BID)A binds tothe defined hydrophobic pocket of BCL-X_(L).

FIGS. 12a and 12b depict the results of studies showing the percent ofcytochrome c released by SAHB3_(BID) compounds from purified mouse livermitochondria.

FIGS. 13a and 13b depict the results of a study showing thatSAHB3_(BID)A- and SAHB3_(BID)B-induced cytochrome c release is fasterand more potent than that of unmodified peptide.

FIG. 14 depicts the results of a study showing that the Gly to Glumutation of SAHB3_(BID)A selectively eliminates Bak-dependent cytochromerelease, underscoring the specificity of action of SAHB3_(BID)A-inducedcytochrome c release shown in FIG. 13.

FIG. 15 depicts the results of a study showing that Jurkat T-cellleukemia cells, upon exposure to FITC-BID BH3 and FITC-BID helix 6, lackfluorescent labeling whereas Jurkat T-cell leukemia cells, upon exposureto FITC-SAHB3_(BID) demonstrate a positive FITC signal, and that theseresults are not significantly altered by trypsin-treatment of the cells.

FIG. 16a depicts the results of a study showing that Jurkat T-cellsexposed to cross-linked peptides FITC-SAHB3_(BID)A and SAHB3_(Bm(G→E))Ademonstrated fluorescent labeling, whereas Jurkat T-cells exposed tounmodified BH3 peptides FITC-BID and FITC-BID_((G→S)) did not.

FIG. 16b depicts the results of a study showing that cellular import ofFITC-SAHB3_(BID)A is time-dependent at 37° C., as assessed by FACSanalysis.

FIGS. 17a and 17b depict the results of a study showing Jurkat T-cellstreated with FITC-peptides at 4° C. and 37° C. FIG. 17a shows thatFITC-BID BH3 did not label the cells at either temperature, andFITC-SAHB3_(BID)A labeled the cells at 37° C. but not 4° C. FIG. 17bshows that FITC-BID helix 6 labels but also permeabilizes the cells in atemperature-independent manner. However, in contrast, FITC-SAHB3_(BID)Aonly labels the cells at 37° C. and does so without cellularpermeabilization, consistent with active transport of SAHB3_(BID)A viaan endocytic pathway.

FIG. 17c depicts the results of a study showing that Jurkat T-cells,when preincubated with or without sodium azide and 2-deoxyglucosefollowed by treatment with FITC-peptides, showed no labeling for eithercondition with the FITC-BID BH3 polypeptide. The cells showed reducedlabeling for FITC-SAHB3_(BID)A under sodium azide and 2-deoxyglucoseconditions, and showed labeling with FITC-BID helix 6 under bothconditions. These results are consistent with an ATP-dependant cellularuptake (e.g., endocytosis pathway) for SAHB3_(BID) import.

FIG. 18 depicts the results of a study showing that FITC-SAHB3_(BID)Auptake is not inhibited by cellular treatment with the glycosaminoglycanheparin, indicating that there are distinctions between the mechanism ofbinding and uptake of FITC-SAHB3_(BID)A compared to other cellpenetrating peptides (CPPs), such as HIV TAT and Antennapedia peptides.

FIGS. 19A and 19B depict the results of a study showing thatFITC-SAHB3BIDA compounds display cytoplasmic labeling with a vesiculardistribution in Jurkat T-cells, whereas plasma membrane fluorescence isnot evident. FIG. 19C indicates that FITC-BID BH3 displays no cellularlabeling of cells. FIG. 19D shows that FITC-BID helix 6 labels the cellsdiffusely and causes significant architectural destruction.

FIGS. 20A-C depict the results of a study showing that FITC-SAHB3BIDAco-localizes with a mitochondrial membrane marker in Jurkat T-cells.FIG. 20A shows the fluorescence corresponding to FITC-SAHB3BIDA peptide.FIG. 20B shows the fluorescence corresponding to the Tom20 mitochondrialmembrane marker. FIG. 20C shows an overlay of FIGS. 20A and 20B.

FIG. 21a and FIG. 21b depicts the results of a study showing thatFITC-SAHB3_(BID)A colocalizes in live BCL-2 overexpressing JurkatT-cells with dextran-labeled endosomes but not transferring-labeledendosomes, indicating that FITC-SAHB3_(BID)A is imported into cells byfluid-phase pinocytosis.

FIG. 21c depicts the results of a study showing that by 24 hours aftertreatment, FITC-SAHB3_(BID)A colocalizes in the live cells withmitochondria labeled by MitoTracker.

FIGS. 22a, 22b, and 22c depict the results of a study showing thatSAHB3_(BID)A triggers metabolic arrest in a dose responsive fashion inthe leukemia cell lines tested, whereas BID BH3 and SAHB3 um_((G→E))Ahad essentially no effect in this dose range.

FIG. 23A depicts the results of a study showing that SAHB3BIDA inducedApoptosis in up to 50% of intact Jurkat cells at 10 μM, an effectspecifically inhibited by BCL-2 overexpression (black bars). FIG. 23Bdepicts the results of a study showing that SAHB3BIDB induced Apoptosisin up to 50% of intact Jurkat cells at 10 μM, an effect specificallyinhibited by BCL-2 overexpression (black bars). Unmodified BID BH3peptide and the gly to glu mutants had no effect based on comparisonwith the no treatment control.

FIG. 24 depicts the results of a study showing the dose response ofJurkat BCL-2 overexpressing cells treated with SAHB3_(BID)A ,SAHB3_(BID(G→E)) and SAHB3_(BID(G→S))A. Whereas SAHB3_(BID)A andSAHB3_(BID(G→S))A can overcome BCL-2 inhibition of apoptosis in thisdose range, the gly to glu point mutant has not effect.

FIG. 25 depicts the results of a study showing that SAHB3_(BID)A treatedleukemia cell lines REH, MV4;11, and SEMK2 underwent specific apoptosisinduction, whereas the gly to glu point mutant SAHB3_(BID(G→E))A had noeffect on the cells.

FIGS. 26a and 26b depict the results of a study showing that bothSAHB3_(BID)A and SAHB_(BID(G→S))A suppressed the growth of SEMK2leukemia in NOD-SCID mice, with SAHB3_(BID(G→S))A demonstrating agreater potency than SAHB3_(BID)A.

FIG. 27a and FIG. 27b depicts the results of a study showing thatSAHB3_(BID)A blunts the progression of SEMK2 leukemia relative tovehicle in NOD-SCID mice. A dose responsive effect is noted in FIG. 27a.

FIGS. 27c, 27d, 27e depict the results of an animal study showing thatSAHB3_(BID)A inhibits the growth of RS4;11 leukemia relative to vehiclein SCID beige mice, with statistically significant prolongation ofsurvival in SAHB3_(BID)A-treated mice compared to vehicle controls.

FIG. 27f depicts the results of an animal study again showing thatSAHB3_(BID)A causes regression of RS4;11 leukemia in SCID beige mice, incontrast with SAHB3_(BID(G→E))A- and vehicle-treated mice whichdemonstrate leukemia progression.

FIGS. 28a-28h depict examples of various alpha helical domains of BCL-2family member proteins (SEQ ID NOs 1-67 and 67-91, respectively, inorder of appearance) amenable to crosslinking.

DETAILED DESCRIPTION

The invention is based, in part, on the discovery that cross-linkedalpha helical domain polypeptides of BCL-2 family proteins have improvedpharmacological properties over their uncrosslinked counterparts (e.g.,increased hydrophobicity, resistance to proteolytic cleavage, bindingaffinity, in vitro and in vivo biological activity). Moreover, it hasbeen surprisingly discovered that the cross-linked polypeptides canpenetrate the cell membrane via a temperature- and energy-dependenttransport mechanism (e.g., endocytosis, specifically fluid-phasepinocytosis). The polypeptides include a tether between two non-naturalamino acids, which tether significantly enhances the alpha helicalsecondary structure of the polypeptide. Generally, the tether extendsacross the length of one or two helical turns (i.e., about 3.4 or about7 amino acids). Accordingly, amino acids positioned at i and i+3; i andi+4; or i and i+7 are ideal candidates for chemical modification andcrosslinking. Thus, for example, where a peptide has the sequence . . .Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉ . . . , crosslinksbetween Xaa_(i) and Xaa₄, or between Xaa_(i) and Xaa₅, or between Xaa₁and Xaa₈ are useful as are crosslinks between Xaa₂ and Xaa₅, or betweenXaa₂ and Xaa₆, or between Xaa₂ and Xaa₉, etc. In addition, a modelpolypeptide was prepared incorporating two sets of crosslinks with onelocated between Xaa₁ and Xaa₅ and another between Xaa₉ and Xaa₁₃. Thedouble crosslink was achieved by careful stereochemical control of thedouble bond metathesis reactions. Thus, the invention encompasses theincorporation of more than one crosslink within the polypeptide sequenceto either further stabilize the sequence or facilitate the stabilizationof longer polypeptide stretches. If the polypeptides are too long to bereadily synthesized in one part, independently synthesized crosslinkedpeptides can be conjoined by a technique called native chemical ligation(Bang, et al., J. Am. Chem Soc. 126:1377).

The novel cross-linked polypeptides are useful, for example, to mimic orstudy proteins or polypeptides having one or more alpha-helical domains.One family of proteins where family members have at least one alphahelical domain is the BCL-2 family of proteins. These proteins areinvolved in cellular apoptotic pathways. Some BCL-2 family members havea pro-apoptotic function, others have an anti-apoptotic function, andstill others change functions with a change in cellular conditions.Accordingly, it is desirable to make stabilized polypeptides that wouldmimic one or more motifs of the BCL-2 family members, thus modulating avariety of BCL-2 related activities.

Chemical Synthesis of a Panel of SAHB3_(BID) Compounds

α,α-Disubstituted non-natural amino acids containing olefinic sidechains of varying length were synthesized according to the schema inFIG. 3 (Williams et al. 1991 J. Am. Chem. Soc. 113:9276; Schafineisteret al. 2000 J. Am. Chem Soc. 122:5891). Chemically crosslinked BID BH3peptides were designed by replacing two or four naturally occurringamino acids with the corresponding synthetic amino acids (FIG. 4a ).Substitutions were made at discrete locations, namely the “i, and i+4positions” or the “i, and i+7 positions”, which facilitate crosslinkingchemistry by placing reactive residues on the same face of the α-helix(FIG. 4b ). Highly conserved amino acids among apoptotic proteins, inaddition to those sequences found to be important in protein-proteininteractions based on X-ray crystallographic and NMR studies (Muchmoreet al. 1996 Nature 381:335; Sattler et al. 1997 Science 275:983), werespecifically not replaced in certain circumstances, conserved aminoacids could be replaced by other amino acids (e.g., syntheticnon-naturally occurring amino acids) to enhance activity (this effectcan be seen in the SAHB3_(BID) mutants described herein). SAHB3_(BID)compounds were generated by solid phase peptide synthesis followed byolefin metathesis-based crosslinking of the synthetic amino acids viatheir olefin-containing side chains. The variations of SAHB3_(BID)compounds generated are illustrated in FIG. 5a . SAHB3_(BID) (SAHB_(A))variants incorporating specific mutations known to alter BID function(Wang et al. 1996 Genes Dev. 10:2859) were also constructed to serve asnegative controls in biological experiments (FIG. 5a ). The aminotermini of selected compounds were further derivatized with fluoresceinisothiocyanate (FITC) or biotin conjugated-lysine to generate labeledSAHB3_(BID) compounds for cell permeability studies and biochemicalassays, respectively (FIG. 5a ). In several syntheses, a C-terminaltryptophan was added to the sequence to serve as a UV label forpurification and concentration determination purposes; the N-terminalglutamic acid was eliminated in several peptides in order to increasethe overall pI of the compound to potentially facilitate cellpenetration (see below). The metathesis approach was readily applied tothe generation of alternate SAHB3s, including SAHB3_(BAD) andSAHB3_(BIM) (FIG. 5a ).

The non-natural amino acids (R and S enantiomers of the 5-carbonolefinic amino acid and the S enantiomer of the 8-carbon olefinic aminoacid) were characterized by nuclear magnetic resonance (NMR)spectroscopy (Varian Mercury 400) and mass spectrometry (Micromass LCT).Peptide synthesis was performed either manually or on an automatedpeptide synthesizer (Applied Biosystems, model 433A), using solid phaseconditions, rink amide AM resin (Novabiochem), and Fmoc main-chainprotecting group chemistry. For the coupling of natural Fmoc-protectedamino acids (Novabiochem), 10 equivalents of amino acid and a 1:1:2molar ratio of coupling reagents HBTU/HOBt (Novabiochem)/DIEA wereemployed. Non-natural amino acids (4 equiv) were coupled with a 1:1:2molar ratio of HATU (Applied Biosystems)/HOBt/DIEA. Olefin metathesiswas performed in the solid phase using 10 mM Grubbs catalyst (Blackewellet al. 1994 supra) (Strem Chemicals) dissolved in degasseddichloromethane and reacted for 2 hours at room temperature. The aminotermini of selected compounds were further derivatized with b-alanineand fluorescein isothiocyanate (FITC [Sigma]/DMF/DIEA) to generatefluorescently labeled compounds. A C-terminal tryptophan wasincorporated to serve as a UV label for purification and concentrationdetermination purposes; SAHBA compounds were also synthesized withoutthe C-terminal tryptophan and N-terminal glutamic acid, the lattermodification performed to increase the overall pI of the molecules.Isolation of metathesized compounds was achieved by trifluoroaceticacid-mediated deprotection and cleavage, ether precipitation to yieldthe crude product, and high performance liquid chromatography (HPLC)(Varian ProStar) on a reverse phase C18 column (Varian) to yield thepure compounds. Chemical composition of the pure products was confirmedby LC/MS mass spectrometry (Micromass LCT interfaced with Agilent 1100HPLC system) and amino acid analysis (Applied Biosystems, model 420A).

FIG. 5b schematically depicts a subset of the peptides in FIG. 5a ,including the stereochemistry of the olefinic amino acids (R and Senantiomers of the 5-carbon olefinic amino acid and the S enantiomer ofthe 8-carbon olefinic amino acid).

SAHB3_(BID) Compounds Display Enhanced α-helicity

We examined the percent helicity of pro-apoptotic BH3 domains, and foundthat these unmodified peptides were predominantly random coils insolution, with α-helical content all under 25% (FIG. 6). Briefly,compounds were dissolved in aqueous 50 mM potassium phosphate solutionpH 7 to concentrations of 25-50 mM. CD spectra were obtained on a JascoJ-710 spectropolarimeter at 20° C. using the following standardmeasurement parameters: wavelength, 190-260 nm; step resolution, 0.5 nm;speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm;path length, 0.1 cm. The α-helical content of each peptide wascalculated by dividing the mean residue ellipticity [φ]222obs by thereported [φ]222obs for a model helical decapeptide (Yang et al. 1986Methods Enzymol. 130:208)).

In each case, the chemical crosslink(s) increased the percent α-helicityof BID's BH3 domain, with SAHB3_(BID)A and B achieving more than 5-foldenhancement (FIG. 7). SAHB3_(BID(G→E))A, a negative control Gly to Glupoint mutant of SAHB3_(BID)A, displays similar helical content toSAHB3_(BID)A (FIG. 8). Thus, the all-hydrocarbon crosslink can transforman apoptotic peptide that is essentially a random coil in aqueoussolution into one that is predominantly α-helical in structure.Interestingly, the importance of the fourth helical turn in stabilizingBID BH3 peptides is underscored by the decrease in helicity observedwhen the SAHB3_(BID)B 23-mer is truncated to the 16-mer, SAHB3_(Bm(tr))B(FIG. 9).

The all-Hydrocarbon Crosslink Increases Protease Resistance ofSAHB3_(BID) Compounds

The amide bond of the peptide backbone is susceptible to hydrolysis byproteases, thereby rendering peptidic compounds vulnerable to rapiddegradation in vivo. Peptide helix formation, however, buries the amidebackbone and therefore shields it from proteolytic cleavage.SAHB3_(BID)A was subjected to in vitro trypsin proteolysis to assess forany change in degradation rate compared to the unmodified BID BH3peptide. SAHB3_(BID)A and unmodified peptide were incubated with trypsinagarose and the reactions quenched at various time points bycentrifugation and subsequent HPLC injection to quantitate the residualsubstrate by ultraviolet absorption at 280 nm. Briefly, BID BH3 andSAHB3_(BID)A compounds (5 mcg) were incubated with trypsin agarose(Pierce) (S/E˜125) for 0, 10, 20, 90, and 180 minutes. Reactions werequenched by tabletop centrifugation at high speed; remaining substratein the isolated supernatant was quantified by HPLC-based peak detectionat 220 nm. The proteolytic reaction displayed first order kinetics andthe rate constant, k, determined from a plot of 1n[S] versus time (k=−1×slope) (FIG. 10a ). The experiment, performed in triplicate,demonstrated a 3.5-fold enhancement in trypsin resistance ofSAHB3_(BID)A compared to the unmodified peptide. Thus, enhancedprotection of trypsin-sensitive amide bonds by burying them at the coreof the α-helix affords a more stable peptidic compound, and maytherefore render such compounds particularly stable in serum.

For ex vivo serum stability studies, FITC-conjugated peptides BID BH3and SAHB3_(BID)A (2.5 mcg) were incubated with fresh mouse serum (20 mL)at 37° C. for 0, 1, 2, 4, 8, and 24 hours. The level of intactFITC-compound was determined by flash freezing the serum specimens inliquid nitrogen, lyophilization, extraction in 50:50 acetonitrile/watercontaining 0.1% trifluoroacetic acid, followed by HPLC-basedquantitation using fluorescence detection at excitation/emissionsettings of 495/530 nm. The results of this analysis are shown in FIG.10 b.

To investigate the in vivo stability of SAHB3_(BID)A, 10 mg/kg ofFITC-conjugated BID

BH3 peptide and SAHB3_(BID)A were injected into NOD-SCID mice and bloodspecimens withdrawn at 0, 1, 4 and 22 hours post-injection. Levels ofintact FITC-compound in 25 μL of fresh serum were then measured. Theresults of this analysis, depicted in FIG. 10c , show that SAHB3_(BID)Awas readily detectable over a 22 hour period, with 13% of the inputstill measurable a 22 hours. In contrast, only 12% of BID BH3 wasdetectable one hour after injection.

SAHB3_(BID) Compounds Retain High Affinity Anti-Apoptotic Binding

The all-hydrocarbon crosslinks were selectively placed on the chargedface of the BID BH3 amphipathic helix in order to avoid interferencewith critical interactions between the binding pocket of multidomainapoptotic proteins and the hydrophobic residues of the BID BH3 helix.Fluorescence polarization competitive binding experiments were performedto evaluate the efficacy of SAHB3_(BID) compounds in competing withFITC-labeled unmodified BID BH3 peptide for GST-BCL-2 binding. AllSAHB3_(BID) compounds demonstrate high affinity binding to GST-BCL2,with SAHB3_(BID)A and B, the two compounds with the greatest percenthelicity, likewise displaying the highest affinity binding (FIG. 11a ).Of note, Gly to Glu mutation of SAHB3_(BID)A and B eliminates highaffinity binding, as would be predicted from previous studies (FIG. 11b). We additionally determined that Gly to Ser mutation of SAHB3_(BID)Aabolishes BCL-2 binding in this assay (data not shown). Truncation ofthe 23-mer SAHB3_(BID)B to a 16-mer results in loss of BCL-2 bindingaffinity, coincident with the decrement in α-helicity described above(FIG. 11c ).

FITC-labeled BID BH3 peptide binds to BCL-2 with a K_(D) of 220 nM, andonce bound, displacement of this interaction by unlabeled BID BH3 occursat an IC₅₀ of 838 nM. This supports a model whereby BH3 binding to BCL-2triggers an overall conformational change favoring the interaction,resulting in the need for excess amounts of unlabeled peptide todisplace prebound FITC-labeled BID BH3. We have further shown that theBAD BH3 domain has an enhanced K_(D) of 41 nM for BCL-2 binding, andthat it can displace prebound FITC-BID BH3 with an IC₅₀ of 173 nM. In asimilar experiment, SAHB3_(BID)A was found to displace FITC-BID BH3 fromBCL-2 with an IC₅₀ of 62 nM, reflecting a more than 13-fold increase indisplacement potency compared to unmodified BID BH3 peptide. These dataconfirm that SAHB3_(BID)A binds with enhanced affinity to BCL-2 comparedto unmodified BH3 peptides, and suggest that preorganization ofα-helical structure by chemical crosslinking provides a kineticadvantage for target binding.

Direct binding assays by fluorescence polarization demonstrated thatincorporation of the crosslink into BID BH3 peptide resulted in enhancedbinding affinity of SAHB3_(BID)A for both BCL-2, an anti-apoptoticmultidomain protein, and BAX, a pro-apoptotic multidomain protein,compared to unmodified BID BH3 peptide (FIGS. 11d and 11e ). A directBCL-2 fluorescence polarization binding assay demonstrated a 6-foldenhancement in BCL-2 binding affinity of SAHB3_(BID)A (K_(D), 38.8 nm)compared to unmodified BID BH3 peptide (K_(D), 269 nM) (FIG. 11d ). AGly to Glu mutation, SAHB_(A(G→E)) (K_(D), 483 nM), eliminates highaffinity binding and serves as a useful control (FIG. 11d ). Briefly,Escherichia coli BL21 (DE3) containing the plasmid encoding C-terminaldeleted GST-BCL-2 were cultured in ampicillin-containing Luria Broth andinduced with 0.1 mM IPTG. The bacterial pellets were resuspended inlysis buffer (1 mg/ml lysozyme, 1% Triton X-100, 0.1 mg/ml PMSF, 2 μg/mlaprotinin, 2 μg/ml leupeptine, 1 μg/ml pepstatin A in PBS) andsonicated. After centrifugation at 20,000×g for 20 min, the supernatantwas applied to a column of glutathione-agarose beads (Sigma). The beadswere washed with PBS and treated with 50 mM glutathione, 50 mM Tris-HCl(pH 8.0) to elute the protein, which was then dialyzed against bindingassay buffer (140 mM NaCl, 50 mM Tris-HCl [pH 7.4]). Fluorescinatedcompounds (25 nM) were incubated with GST-BCL2 (25 nM-1000 nM) inbinding buffer at room temperature. Binding activity was measured byfluorescence polarization on a Perkin-Elmer LS50B luminescencespectrophotometer. KD values were determined by nonlinear regressionanalysis using Prism software (Graphpad). Full-length BAX protein wasprepared as previously described (Suzuki et al, Cell, 103:645) andfluorescence polarization assay performed as described above.

SAHB3_(BID)A Binds to BCL-X_(L)

To determine if SAHB3_(BID)A specifically interacts with the definedbinding groove of an anti-apoptotic multidomain protein, atwo-dimensional ¹⁵N/¹H heteronuclear single-quantum correlation (HSQC)spectrum of ¹⁵N-labeled BCL-X_(L) before and after the addition ofSAHB3_(BID)A was recorded and compared with the corresponding BIDBH3/¹⁵N—BCL-X_(L) spectrum. Briefly, Escherichia coli BL21 (DE3)containing the plasmid encoding C-terminal deleted BCL-X_(L) werecultured in M9-minimal medium containing ¹⁵NH₄Cl (Cambridge IsotopeLaboratories) to generate uniformly ¹⁵N-labeled protein. Recombinantproteins were isolated from bacteria. Unlabeled SAHB3_(BID)A and BID BH3peptides were generated and purified as described above. The following1:1 complexes were prepared at 0.1 mM in 50 mM potassium phosphate (pH7), 50 mM sodium chloride, 5% DMSO in D₂O or H₂O/D₂O (95:5):¹⁵N—BCL-X_(L)/unlabeled BID BH3, ¹⁵N—BCL-X_(L)/unlabeled SAHB3_(BID)A.Two dimensional ¹⁵N/¹H heteronuclear single-quantum spectra wererecorded for the two complexes and analyzed for changes in resonanceupon ligand binding.

The overall similarity of the HSQC spectra indicates that the structuralchanges occurring in BCL-X_(L) after addition of SAHB3_(BID)A are nearlyidentical to those observed with BID BH3 peptide (FIG. 11f ).

SAHB3_(BID) Compounds Trigger Rapid and Specific Release ofMitochondrial Cytochrome C

In order to assess the biological activity of SAHB3BID compounds invitro, cytochrome c release assays were performed using purified mouseliver mitochondria. Mitochondria (0.5 mg/mL) were incubated for 40minutes with 1 μM and 100 nM of SAHB3_(BID) compounds and thensupernatants and mitochondrial fractions isolated and subjected tocytochrome c ELISA assay. Background cytochrome c release (10-15%) wassubtracted from total release for each sample, and the percent actualcytochrome c release was determined (FIG. 12). The identical experimentwas performed concurrently on mouse liver mitochondria isolated fromBak−/− mice, which do not release mitochondrial cytochrome c in responseto BID-BH3 activation; data from the BAK−/− mitochondria therefore serveas a negative control for BAK-mediated cytochrome c release in responseto SAHB3_(BID) treatments. In each case, except for the doublecross-linked SAHB3_(BID)E (which may lack critical amino acids forbiological activity or, in this case, be overly constrained by the dualcross-links), there is approximately a doubling of cytochrome c releasein response to 1 μM SAHB3_(BID) compounds compared to the unmodifiedpeptide (FIG. 12a ). BAK-independent cytochrome c release is observed atthis dose with SAHB3_(BID)A, B, and, in particular, D. Whereas thiscytochrome c release may represent a nonspecific membrane perturbingeffect of the α-helices, the role of a SAHB3_(BID)-induced,BAK-independent component of cytochrome c release is worthy of furtherexploration. Interestingly, the SAHB3_(BID) compound that induces themost significant level of BAK-independent cytochrome c release,SAHB3_(BID)D, is also the most hydrophobic of the SAHB3_(BID) compounds;SAHB3_(BID)D elutes from the reverse phase C18 column at 95%acetonitrile/5% water, compared to the other SAHB3BID compounds thatelute at 50-75% acetonitrile. BID mutants with defective BH3 domains canpromote BAK-independent cytochrome c mobilization (Scorrano et al, DevCell, 2:55), and the highly hydrophobic BID helix 6 has been implicatedin this activity (L. Scorrano, S. J. Korsmeyer, unpublished results). Itis plausible that SAHB3_(BID)D displays both BAK dependent andindependent cytochrome c release by mimicking features of BID helices 3and 6. At ten-fold lower dosing SAHB3_(BID)A and B retain selectiveBAK-dependent cytochrome c release activity (FIG. 12b ). The potency ofSAHB3_(BID)B, in particular, compares favorably with maximally activatedmyristolated BID protein, which releases approximately 65% cytochrome cunder these conditions at doses of 30 nM.

The most active SAHB3_(BID) compounds, A and B, were subjected tofurther kinetic studies to determine if helical preorganization cantrigger more rapid cytochrome c release compared to the unmodifiedpeptide. Similar to the above experiment, mouse liver mitochondria fromwild-type and Bak−/− mice were exposed to the compounds at variousconcentrations and assayed for cytochrome c release at 10 and 40 minuteintervals. Whereas at 10 minutes the unmodified peptide causes less than10% release at the highest dose tested (1 μM), SAHB3_(BID)B has an EC50for release at this timepoint of just under 400 nM, with almost maximalcytochrome release at 1 μM (FIG. 13a ). Likewise, SAHB3_(BID)A triggerssignificant cytochrome c release at the 10 minute time interval. TheEC50 for cytochrome c release at 40 minutes is 2.9 μM for the unmodifiedpeptide and 310 and 110 nM for SAHB3_(BID) A and B, respectively (FIG.13b ). Thus, SAHB3_(BID)A and B display a 10-25 fold enhancement incytochrome c release activity at the 40 minute time point. Whereas theBAK-dependent cytochrome c release increases over time, theBAK-independent release does not change between the 10 and 40 minutetimepoints, suggesting that this distinct release occurs early and ismaximally achieved within 10 minutes. Of note, the negative control Glyto Glu point mutant of SAHB3_(BID)A, SAHB3_(BID(G→E))A, generates onlyBak-independent cytochrome c release, confirming that SAHB3_(BID)Afunctions via the Bak-dependent mitochondrial apoptosis pathway (FIG.14). Taken together, these cytochrome c release data indicate thatSAHB3_(BID)A and B are capable of specifically inducing BAK-dependentcytochrome c release with markedly enhanced potency and kineticscompared to the unmodified peptide.

SAHB3_(BID) Compounds Penetrate Intact Cells

Fluorescein-derivatized SAHB3_(BID) compounds, BID BH3 peptides, and aBID helix 6 peptide were incubated with Jurkat T-cell leukemia cells inculture for 4-24 hours and subsequently FACS sorted to determine percentlabeling of leukemia cells. In order to avoid confounding results fromcell-surface bound compounds, the Jurkat cells were washed thoroughlyand subjected to trypsin overdigestion, in accordance with recentreports. For each compound tested, there was no significant change inthe FITC signal profile after trypsin digestion, suggesting that in thecase of these peptides, little to no FITC-labeled compound is surfacebound (FIG. 15). Whereas BID BH3-treated cells were FITC-negative, bothFITC-SAHB3_(BID)A- and FITC-SAHB3_(BID(G→E))A-treated cells wereFITC-positive, as indicated by a rightward shift of the FITC signal(FIG. 16a ). The similar profile of FITC-SAHB3_(BID)A andFITC-SAHB3_(BID(G→E))A in these cell permeability studies isparticularly important, given the use of the point mutant compound as anegative control in biological experiments. BID helix 6, a cellpermeable and membrane perturbing peptide, was used as a positivecontrol for FITC-labeling in this experiment.

Surprisingly, it was discovered that FITC-SAHB3_(BID)A appears to enterthe cell via endocytosis, a temperature- and energy-dependent transportpathway. Cellular import of FITC-SAHB3_(BID)A occurred in atime-dependent manner (FIG. 16b ). When cellular endocytosis wasinhibited by performing the experiment at 4° C. (FIG. 17a, 17b ) or bytreatment with the energy poisons sodium azide and 2-deoxyglucose (FIG.17c ), cell labeling was inhibited or markedly diminished, respectively.Of note, Jurkat cells labeled by FITC-SAHB3_(BID)A at 37° C. arepropidium iodide (PI) negative, confirming that the crosslinked peptidedoes not merely function as a permeabilizing agent (FIG. 17b ); incontrast, FITC-BID helix 6 readily penetrates at both temperatures,effectively permeabilizing the cells, as evidenced by the degree of PIpositivity (FIG. 17b ). These data support an endocytic mechanism ofentry for the SAHB3_(BID) compounds, consistent with recent reportsciting cell-surface adherence followed by endocytosis as the mechanismof entry for other cell-penetrating peptides (CPPs), such as the HIVtransactivator of transcription (TAT). Whereas highly basic CPPs, suchas TAT and Antennapedia, are believed to be concentrated at the cellsurface by adherence to negatively charged glycosaminoglycansSAHB3_(BID)A import was not inhibited in a dose-responsive manner byheparin (FIG. 18) The biophysical properties of the SAHB3_(BID)amphipathic α-helix may facilitate distinct cell contacts viaelectrostatic and/or lipid membrane interactions.

Confocal microscopy experiments were employed in order to determine theintracellular localization of SAHB3_(BID)A. Jurkat T-cell leukemia cellswere incubated with FITC-labeled compounds as described above or withserum replacement at 4 hours followed by additional 16 hours incubationat 37° C., and after washing twice with PBS, were cytospun at 600 RPMfor 5 minutes onto superfrost plus glass slides (Fisher). Cells werethen fixed in 4% paraformaldehyde, washed with PBS, incubated withTO-PRO-3 iodide (100 nM) (Molecular Probes) to counterstain nuclei,treated with Vectashield mounting medium (Vector), and then imaged byconfocal microscopy (BioRad 1024). For double labeling experiments,fixed cells were additionally incubated with primary antibody to TOM20,and rhodamine-conjugated secondary antibody prior to TOPRO-3counterstaining. For live confocal microscopy, double labeling of Jurkatcells was performed with FITC-SAHB_(A) (10 μM) and MitoTracker (100 nM,Molecular Probes), tetramethylrhodamine isothiocyanate (TRITC)-Dextran4.4 kD or 70 kD (25 mcg/mL, Molecular Probes), or Alexa Fluor594-transferrin (25 mcg/mL, Molecular Probes) for 4 hours (dextran andtransferrin) or 24 hours (MitoTracker). Due to limitations ofphotobleaching, BCL-2 overexpressing Jurkat cells were used for liveconfocal microscopy in order to optimize FITC imaging. FITC-SAHB_(A)labeling of mitochondria was brighter in BCL-2 overexpressing Jurkats(consistent with the mechanism for SAHB activity), and thus imagecapture was facilitated using these cells. Treated Jurkats were washedtwice and then resuspended in PBS and wet mount preparations analyzedwith a BioRad 1024 (Beth Israel/Deaconess Center for AdvancedMicroscopy) or Zeiss LSM510 laser scanning confocal microscope(Children's Hospital Boston Imaging Core).

In fixed sections, SAHB3_(BID)A compounds localized to the cytoplasmicrim of the leukemic cells, with no plasma membrane or surfacefluorescence evident; the vesicular pattern of fluorescence suggested anorganelle-specific localization (FIGS. 19a and 19b ). Consistent withthe FACS data, Jurkat cells treated with FITC-BID BH3 showed nofluorescent labeling (FIG. 19c ). Whereas FITC-SAHB3_(BID)A-treatedcells display selective intracellular fluorescence and maintain theircellular architecture (FIG. 19a ), FITC-BID helix 6-treated cells arediffusely labeled and demonstrate disrupted cellular morphology (FIGS.19d ). Colocalization studies using FITC-SAHB3_(BID)A and an antibody tomitochondrial membrane protein Tom20, demonstrated extensive overlap ofSAHB3_(BID)A fluorescence with mitochondria, the expected site ofSAHB3_(BID)'s molecular targets (FIG. 20).

Live cell imaging performed 4 hr after SAHB treatment demonstrated aninitial colocalization of FITC-SAHB_(A) with dextran (4.4 kD or 70kD)-labeled endosomes (FIG. 21a ), but not transferrin-labeled endosomes(FIG. 21b ), consistent with cellular uptake by fluid-phase pinocytosis(manuscript ref 27), the endocytic pathway determined for TAT and Antppeptides (manuscript ref 28). At a 24 hr time point, intracellularFITC-SAHB_(A) showed increased colocalization with MitoTracker-labeledmitochondria in live cells (FIG. 21c ) consistent with the mitochondrialcolocalization observed in fixed cells using an antibody to Tom20, amitochondrial outer membrane protein (FIG. 20). Taken together, the FACSdata and confocal imaging demonstrate that the all-hydrocarbon crosslinkenables SAHB3_(BID)A compounds to be imported by intact cells (e.g.,through an endocytotic mechanism).

SAHB3_(BID) Compounds Trigger Apoptosis of B-, T-, and Mixed-LineageLeukemia (MLL) Cells

In order to assess whether SAHB3_(BID) compounds could arrest the growthof proliferating leukemia cells in culture,3-(4,5-dimethylthiazol-2-yl)2,5-dipheny tetrazolium bromide, MTT assaysusing serial dilutions of SAHB3_(BID)A were performed on T-cell(Jurkat), B-cell (REH), and Mixed Lineage Leukemia (MLL)-cells (MV4;11,SEMK2, RS4;11) in culture. SAHB3_(BID)A inhibited the leukemic cells atIC₅₀s of 2.2 (Jurkat), 10.2 (REH), 4.7 (MV4;11), 1.6 (SEMK2), and 2.7(RS4;11) μM (FIG. 22a ). Neither the BID BH3 peptide nor theSAHB_(A(G→E)) point mutant had an effect in this dose range (FIG. 22b,22c ).

To assess whether this metabolic arrest represented apoptosis induction,Jurkat leukemia cells were treated with 10 μM SAHB3_(BID)A and B,SAHB3_(BID(G→E))A and B, and unmodified BID BH3 peptide, in serum-freemedia for 4 hours followed by a 16 hour incubation in serum-containingmedia (ie. final peptide concentrations of 5 μM), and then assayed forapoptosis by flow cytometric detection of annexin V-treated cells.SAHB3_(BID)A and B demonstrated between 40-60% annexin V positivity by20 hours post treatment, whereas the unmodified peptide and SAHB3_(BID)point mutants had no effect (FIGS. 23a and 23b ). Comparable studiesthat use either unmodified BH3 peptides with carrier reagents orengineered helices with nonspecific mitochondrial perturbing effects,required doses of 200-300 μM to activate apoptosis. An additionalcontrol experiment using Jurkat cells engineered to overexpress BCL-2was subsequently undertaken to assess whether SAHB3_(BID)-inducedapoptosis could be decreased by excess BCL-2, which would suggest thatthe compounds specifically function within cells through themitochondrial apoptosis pathway. Indeed, the pro-apoptotic effect of 10uM SAHB3_(BID)A and B on “wild-type” Jurkats was abolished in the BCL-2overexpressing cells. This protective effect, however, can be overcomeby dose escalation of SAHB3_(BID)A but not of SAHB3_(BID(G→E))A (FIG.24); in addition, a gly to ser point mutant of SAHB3_(BID)A(SAHB3_(BID(G→S))A), which does not exhibit BCL-2 binding affinity (seeabove), is equally effective as a pro-apoptotic in “wild-type” and BCL-2overexpressing Jurkat cells (FIG. 24). Apoptosis induction assays usingSAHB3_(BID)A and SAHB3_(BID(G→E))A were additionally performed in theREH, MV4;11, and SEMK2 cell lines with similar results (FIG. 25). Takentogether, these data indicate that SAHB3_(BID) compounds can penetrateand kill proliferating leukemia cells. The observed pro-apoptoticeffects are selectively abolished by gly to glu mutation of SAHB3_(BID)Aand cellular overexpression of BCL-2, findings which underscore thatSAHB3_(BID) compounds function via the defined mitochondrial apoptosispathway.

SAHB3_(BID)A and SAHB3_(BIDG→S)A Demonstrate Leukemic Suppression invivo

NOD-SCID mice were subjected to 300 cGy total body irradiation followedby intravenous injection of 4×10⁶ SEMK2-M1 leukemia cells exhibitingstable luciferase expression. The mice were monitored weekly forleukemia engraftment using the In Vivo Imaging System (IVIS, Xenogen),which quantitates total body luminescence after intraperitonealinjection of D-luciferin. On day 0, the leukemic mice were imaged andthen treated intravenously with 10 mg/kg of SAHB3_(BID)A,SAHB3_(BIDG-->s)A, or no injection on days 1, 2, 3, 5, 6. Total bodyluminescence was measured on days 4 and 7. Referring to FIG. 26a ,analysis of tumor burden among the groups demonstrates leukemicsuppression by SAHB3_(BID)A and SAHB3_(BID(G-->s))A compared tountreated control mice. Referring to FIG. 26b , total body luminescenceimages demonstrate more advanced leukemia in the untreated group by day7 (red density, representing high level leukemia, is seen throughout theskeletal system) compared to the SAHB3_(BID)A-treated mice, whichdemonstrate lower level and more localized disease. Interestingly, theG-->S mutant, which cannot be sequestered by BCL-2 appears to be morepotent than the parent compound, SAHB3_(BID)A, in suppressing leukemicgrowth.

In further animal experiments, leukemic mice (generated as above) wereimaged on day 0 and then treated intravenously with 10 mg/kgSAHB3_(BID)A, 5 mg/kg SAHB3_(BID)A, or vehicle control (5% DMSO in D5W)on days 1, 2, 3, 6, and 7. Total body luminescence was measured on days4 and 8. Referring to FIG. 27a , analysis of tumor burden among thegroups demonstrates leukemic suppression by SAHB3_(BID)A in adose-dependent manner compared to untreated control mice. Referring toFIG. 27b , total body luminescence images demonstrate more advancedleukemia in the untreated group by day 8 (red density represents highlevel leukemia) compared to the SAHB3_(BID)A-treated mice, whoseleukemic progression is noticeably blunted.

In additional animal experiments that instead employed SCID beige miceand RS4;11 leukemia cells, SAHB3_(BID)A treatment consistentlysuppressed leukemia growth in vivo. For in vivo leukemia imaging, micewere anesthetized with inhaled isoflurane (Abbott Laboratories) andtreated concomitantly with intraperitoneal injection of D-luciferin (60mg/kg) (Promega). Photonic emission was imaged (2 min exposure) usingthe In Vivo Imaging System (Xenogen) and total body bioluminescencequantified by integration of photonic flux (photons/sec) (Living ImageSoftware, Xenogen). Starting on experimental day 1, mice received adaily tail vein injection of SAHB3_(BID)A (10 mg/kg) or vehicle (5% DMSOin D5W) for seven days. Mice were imaged on days 1, 3, and 5 andsurvival monitored daily for the duration of the experiment. Thesurvival distributions of SAHB3_(BID)A and vehicle-treated mice weredetermined using the Kaplan-Meier method and compared using the log-ranktest. The Fisher's Exact test was used to compare the proportion of micewho failed treatment between days 3 and 5, where treatment failure wasdefined as progression or death, and success as stable disease orregression. Expired mice were subjected to necropsy (RodentHistopathology Core, DF/HCC).

Control mice demonstrated progressive acceleration of leukemic growth asquantitated by increased bioluminescent flux from days 1-5 (FIG. 27c ).SAHB3_(BID)A treatment suppressed the leukemic expansion after day 3,with tumor regression observed by day 5. Representative mouse imagesdemonstrate the progressive leukemic infiltration of spleen and liver inmice, but regression of disease at these anatomical sites inSAHB_(A)-treated mice by day 5 of treatment (FIG. 27d ). The median timeto death in this cohort was 5 days for control animals, whereas none ofthe SAHB_(A)-treated animals dies during the seven day treatment period,and instead survived for a median of 11 days (FIG. 27e ). Histologicexamination of SAHB_(A)-treated mice showed no obvious toxicity of thecompound to normal tissue. In an additional study comparingSAHB3_(BID)A- and SAHB3_(BID(G→E))A-treated mice, animals receiving thepoint mutant SAHB did not exhibit tumor regression (FIG. 27f ),highlighting the in vivo specificity of SAHB3_(BID)A's anti-leukemicactivity.

Polypeptides

In some instances, the hydrocarbon tethers (i.e., cross links) describedherein can be further manipulated. In one instance, a double bond of ahydrocarbon alkenyl tether, (e.g., as synthesized using aruthenium-catalyzed ring closing metathesis (RCM)) can be oxidized(e.g., via epoxidation or dihydroxylation) to provide one of compoundsbelow.

Either the epoxide moiety or one of the free hydroxyl moieties can befurther functionalized. For example, the epoxide can be treated with anucleophile, which provides additional functionality that can be used,for example, to attach a tag (e.g., a radioisotope or fluorescent tag).The tag can be used to help direct the compound to a desired location inthe body (e.g., directing the compound to the thyroid when using anIodine tag) or track the location of the compound in the body.Alternatively, an additional therapeutic agent can be chemicallyattached to the functionalized tether (e.g., an anti-cancer agent suchas rapamycin, vinblastine, taxol, etc.). Such derivitization canalternatively be achieved by synthetic manipulation of the amino orcarboxy terminus of the polypeptide or via the amino acid side chain.

While hydrocarbon tethers have been described, other tethers are alsoenvisioned. For example, the tether can include one or more of an ether,thioether, ester, amine, or amide moiety. In some cases, a naturallyoccurring amino acid side chain can be incorporated into the tether. Forexample, a tether can be coupled with a functional group such as thehydroxyl in serine, the thiol in cysteine, the primary amine in lysine,the acid in aspartate or glutamate, or the amide in asparagine orglutamine. Accordingly, it is possible to create a tether usingnaturally occurring amino acids rather than using a tether that is madeby coupling two non-naturally occurring amino acids. It is also possibleto use a single non-naturally occurring amino acid together with anaturally occurring amino acid.

It is further envisioned that the length of the tether can be varied.For instance, a shorter length of tether can be used where it isdesirable to provide a relatively high degree of constraint on thesecondary alpha-helical structure, whereas, in some instances, it isdesirable to provide less constraint on the secondary alpha-helicalstructure, and thus a longer tether may be desired.

Additionally, while examples of tethers spanning from amino acids i toi+3, i to i+4; and i to i+7 have been described in order to provide atether that is primarily on a single face of the alpha helix, thetethers can be synthesized to span any combinations of numbers of aminoacids.

In some instances, alpha disubstituted amino acids are used in thepolypeptide to improve the stability of the alpha helical secondarystructure. However, alpha disubstituted amino acids are not required,and instances using mono-alpha substituents (e.g., in the tethered aminoacids) are also envisioned.

As can be appreciated by the skilled artisan, methods of synthesizingthe compounds of the described herein will be evident to those ofordinary skill in the art. Additionally, the various synthetic steps maybe performed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The peptides of this invention can be made by chemical synthesismethods, which are well known to the ordinarily skilled artisan. See,for example, Fields et al., Chapter 3 in Synthetic Peptides: A User'sGuide, ed. Grant, W. H. Freeman & Co., New York, N.Y., 1992, p. 77.Hence, peptides can be synthesized using the automated Merrifieldtechniques of solid phase synthesis with the α-NH₂ protected by eithert-Boc or F-moc chemistry using side chain protected amino acids on, forexample, an Applied Biosystems Peptide Synthesizer Model 430A or 431.

One manner of making of the peptides described herein is using solidphase peptide synthesis (SPPS). The C-terminal amino acid is attached toa cross-linked polystyrene resin via an acid labile bond with a linkermolecule. This resin is insoluble in the solvents used for synthesis,making it relatively simple and fast to wash away excess reagents andby-products. The N-terminus is protected with the Fmoc group, which isstable in acid, but removable by base. Any side chain functional groupsare protected with base stable, acid labile groups.

Longer peptides could be made by conjoining individual syntheticpeptides using native chemical ligation. Alternatively, the longersynthetic peptides can be synthesized by well known recombinant DNAtechniques. Such techniques are provided in well-known standard manualswith detailed protocols. To construct a gene encoding a peptide of thisinvention, the amino acid sequence is reverse translated to obtain anucleic acid sequence encoding the amino acid sequence, preferably withcodons that are optimum for the organism in which the gene is to beexpressed. Next, a synthetic gene is made, typically by synthesizingoligonucleotides which encode the peptide and any regulatory elements,if necessary. The synthetic gene is inserted in a suitable cloningvector and transfected into a host cell. The peptide is then expressedunder suitable conditions appropriate for the selected expression systemand host. The peptide is purified and characterized by standard methods.

The peptides can be made in a high-throughput, combinatorial fashion,e.g., using a high-throughput polychannel combinatorial synthesizeravailable from Advanced Chemtech.

FIGS. 28a-28f depict various peptides that include domains that areuseful for creating cross-linked peptides.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant (e.g., insufficient orexcessive) BCL-2 family member expression or activity (e.g., extrinsicor intrinsic apoptotic pathway abnormalities). As used herein, the term“treatment” is defined as the application or administration of atherapeutic agent to a patient, or application or administration of atherapeutic agent to an isolated tissue or cell line from a patient, whohas a disease, a symptom of disease or a predisposition toward adisease, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve or affect the disease, the symptoms ofdisease or the predisposition toward disease. A therapeutic agentincludes, but is not limited to, small molecules, peptides, antibodies,ribozymes and antisense oligonucleotides.

It is possible that some BCL-2 type disorders can be caused, at least inpart, by an abnormal level of one or more BCL-2 family members (e.g.,over or under expression), or by the presence of one or more BCL-2family members exhibiting abnormal activity. As such, the reduction inthe level and/or activity of the BCL-2 family member or the enhancementof the level and/or activity of the BCL-2 family member, which wouldbring about the amelioration of disorder symptoms.

The polypeptides of the invention can be used to treat, prevent, and/ordiagnose cancers and neoplastic conditions. As used herein, the terms“cancer”, “hyperproliferative” and “neoplastic” refer to cells havingthe capacity for autonomous growth, i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth. Hyperproliferativeand neoplastic disease states may be categorized as pathologic, i.e.,characterizing or constituting a disease state, or may be categorized asnon-pathologic, i.e., a deviation from normal but not associated with adisease state. The term is meant to include all types of cancerousgrowths or oncogenic processes, metastatic tissues or malignantlytransformed cells, tissues, or organs, irrespective of histopathologictype or stage of invasiveness. “Pathologic hyperproliferative” cellsoccur in disease states characterized by malignant tumor growth.Examples of non-pathologic hyperproliferative cells includeproliferation of cells associated with wound repair.

Examples of cellular proliferative and/or differentiative disordersinclude cancer, e.g., carcinoma, sarcoma, or metastatic disorders. Thecompounds (i.e., polypeptides) can act as novel therapeutic agents forcontrolling breast cancer, ovarian cancer, colon cancer, lung cancer,metastasis of such cancers and the like. A metastatic tumor can arisefrom a multitude of primary tumor types, including but not limited tothose of breast, lung, liver, colon and ovarian origin.

Examples of cancers or neoplastic conditions include, but are notlimited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer,esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer,prostate cancer, uterine cancer, cancer of the head and neck, skincancer, brain cancer, squamous cell carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicularcancer, small cell lung carcinoma, non-small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposisarcoma.

Examples of proliferative disorders include hematopoietic neoplasticdisorders. As used herein, the term “hematopoietic neoplastic disorders”includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias, e.g., erythroblasticleukemia and acute megakaryoblastic leukemia. Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit. Rev. inOncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

Examples of cellular proliferative and/or differentiative disorders ofthe breast include, but are not limited to, proliferative breast diseaseincluding, e.g., epithelial hyperplasia, sclerosing adenosis, and smallduct papillomas; tumors, e.g., stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma,invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)carcinoma, tubular carcinoma, and invasive papillary carcinoma, andmiscellaneous malignant neoplasms. Disorders in the male breast include,but are not limited to, gynecomastia and carcinoma.

Examples of cellular proliferative and/or differentiative disorders ofthe lung include, but are not limited to, bronchogenic carcinoma,including paraneoplastic syndromes, bronchioloalveolar carcinoma,neuroendocrine tumors, such as bronchial carcinoid, miscellaneoustumors, and metastatic tumors; pathologies of the pleura, includinginflammatory pleural effusions, noninflammatory pleural effusions,pneumothorax, and pleural tumors, including solitary fibrous tumors(pleural fibroma) and malignant mesothelioma.

Examples of cellular proliferative and/or differentiative disorders ofthe colon include, but are not limited to, non-neoplastic polyps,adenomas, familial syndromes, colorectal carcinogenesis, colorectalcarcinoma, and carcinoid tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe liver include, but are not limited to, nodular hyperplasias,adenomas, and malignant tumors, including primary carcinoma of the liverand metastatic tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe ovary include, but are not limited to, ovarian tumors such as,tumors of coelomic epithelium, serous tumors, mucinous tumors,endometeriod tumors, clear cell adenocarcinoma, cystadenofibroma,brenner tumor, surface epithelial tumors; germ cell tumors such asmature (benign) teratomas, monodermal teratomas, immature malignantteratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sexcord-stomal tumors such as, granulosa-theca cell tumors,thecoma-fibromas, androblastomas, hill cell tumors, and gonadoblastoma;and metastatic tumors such as Krukenberg tumors.

The polypeptides described herein can also be used to treat, prevent ordiagnose conditions characterised by overactive cell death or cellulardeath due to physiologic insult etc. Some examples of conditionscharacterized by premature or unwanted cell deth are or alternativelyunwanted or excessive cellular proliferation include, but are notlimited to hypocellular/hypoplastic, acellular/aplastic, orhypercellular/hyperplastic conditions. Some examples include hematologicdisorders including but not limited to fanconi anemia, aplastic anemia,thalaessemia, congenital neutropenia, myelodysplasia.

The polypeptides of the invention that act to decrease apoptosis can beused to treat disorders associated with an undesirable level of celldeath. Thus, the anti-apoptotic peptides of the invention can be used totreat disorders such as those that lead to cell death associated withviral infection, e.g., infection associated with infection with humanimmunodeficiency virus (HIV). A wide variety of neurological diseasesare characterized by the gradual loss of specific sets of neurons, andthe anti-apoptotic peptides of the infection can be used in thetreatment of these disorders. Such disorders include Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis (ALS)retinitis pigmentosa, spinal muscular atrophy, and various forms ofcerebellar degeneration. The cell loss in these diseases does not inducean inflammatory response, and apoptosis appears to be the mechanism ofcell death. In addition, a number of hematologic diseases are associatedwith a decreased production of blood cells. These disorders includeanemia associated with chronic disease, aplastic anemia, chronicneutropenia, and the myelodysplastic syndromes. Disorders of blood cellproduction, such as myelodysplastic syndrome and some forms of aplasticanemia, are associated with increased apoptotic cell death within thebone marrow. These disorders could result from the activation of genesthat promote apoptosis, acquired deficiencies in stromal cells orhematopoietic survival factors, or the direct effects of toxins andmediators of immune responses. Two common disorders associated with celldeath are myocardial infarctions and stroke. In both disorders, cellswithin the central area of ischemia, which is produced in the event ofacute loss of blood flow, appear to die rapidly as a result of necrosis.However, outside the central ischemic zone, cells die over a moreprotracted time period and morphologically appear to die by apoptosis.The anti-apoptotic peptides of the invention can be used to treat allsuch disorders associated with undesirable cell death.

Some examples of immunologic disorders that can be treated with thepolypeptides described herein include but are not limited to organtransplant rejection, arthritis, lupus, IBD, Crohn's Disease, asthma,multiple sclerosis, diabetes etc.

Some examples of neurologic disorders that can be treated with thepolypeptides described herein include but are not limited to Alzheimer'sDisease, Down's Syndrome, Dutch Type Hereditary Cerebral HemorrhageAmyloidosis, Reactive Amyloidosis, Familial Amyloid Nephropathy withUrticaria and Deafness, Muckle-Wells Syndrome, Idiopathic Myeloma;Macroglobulinemia-Associated Myeloma, Familial Amyloid Polyneuropathy,Familial Amyloid Cardiomyopathy, Isolated Cardiac Amyloid, SystemicSenile Amyloidosis, Adult Onset Diabetes, Insulinoma, Isolated AtrialAmyloid, Medullary Carcinoma of the Thyroid, Familial Amyloidosis,Hereditary Cerebral Hemorrhage With Amyloidosis, Familial AmyloidoticPolyneuropathy, Scrapie, Creutzfeldt-Jacob Disease, GerstmannStraussler-Scheinker Syndrome, Bovine Spongiform Encephalitis, aPrion-mediated disease, and Huntington's Disease.

Some examples of endocrinologic disorders that can be treated with thepolypeptides described herein include but are not limited to diabetes,hypthyroidism, hyopituitarism, hypoparathyroidism, hypogonadism, etc.

Examples of cardiovascular disorders (e.g., inflammatory disorders) thatcan be treated or prevented with the compounds and methods of theinvention include, but are not limited to, atherosclerosis, myocardialinfarction, stroke, thrombosis, aneurism, heart failure, ischemic heartdisease, angina pectoris, sudden cardiac death, hypertensive heartdisease; non-coronary vessel disease, such as arteriolosclerosis, smallvessel disease, nephropathy, hypertriglyceridemia, hypercholesterolemia,hyperlipidemia, xanthomatosis, asthma, hypertension, emphysema andchronic pulmonary disease; or a cardiovascular condition associated withinterventional procedures (“procedural vascular trauma”), such asrestenosis following angioplasty, placement of a shunt, stent, syntheticor natural excision grafts, indwelling catheter, valve or otherimplantable devices. Preferred cardiovascular disorders includeatherosclerosis, myocardial infarction, aneurism, and stroke.

Pharmaceutical Compositions and Routes of Administration

As used herein, the compounds of this invention, including the compoundsof formulae described herein, are defined to include pharmaceuticallyacceptable derivatives or prodrugs thereof. A “pharmaceuticallyacceptable derivative or prodrug” means any pharmaceutically acceptablesalt, ester, salt of an ester, or other derivative of a compound of thisinvention which, upon administration to a recipient, is capable ofproviding (directly or indirectly) a compound of this invention.Particularly favored derivatives and prodrugs are those that increasethe bioavailability of the compounds of this invention when suchcompounds are administered to a mammal (e.g., by allowing an orallyadministered compound to be more readily absorbed into the blood) orwhich enhance delivery of the parent compound to a biologicalcompartment (e.g., the brain or lymphatic system) relative to the parentspecies. Preferred prodrugs include derivatives where a group whichenhances aqueous solubility or active transport through the gut membraneis appended to the structure of formulae described herein.

The compounds of this invention may be modified by appending appropriatefunctionalities to enhance selective biological properties. Suchmodifications are known in the art and include those which increasebiological penetration into a given biological compartment (e.g., blood,lymphatic system, central nervous system), increase oral availability,increase solubility to allow administration by injection, altermetabolism and alter rate of excretion.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, benzoate, benzenesulfonate, butyrate, citrate,digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate,heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate,nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate,salicylate, succinate, sulfate, tartrate, tosylate and undecanoate.Salts derived from appropriate bases include alkali metal (e.g.,sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄⁺ salts. This invention also envisions the quaternization of any basicnitrogen-containing groups of the compounds disclosed herein. Water oroil-soluble or dispersible products may be obtained by suchquaternization.

The compounds of the formulae described herein can, for example, beadministered by injection, intravenously, intraarterially, subdermally,intraperitoneally, intramuscularly, or subcutaneously; or orally,buccally, nasally, transmucosally, topically, in an ophthalmicpreparation, or by inhalation, with a dosage ranging from about 0.001 toabout 100 mg/kg of body weight, or according to the requirements of theparticular drug. The methods herein contemplate administration of aneffective amount of compound or compound composition to achieve thedesired or stated effect. Typically, the pharmaceutical compositions ofthis invention will be administered from about 1 to about 6 times perday or alternatively, as a continuous infusion. Such administration canbe used as a chronic or acute therapy. The amount of active ingredientthat may be combined with the carrier materials to produce a singledosage form will vary depending upon the host treated and the particularmode of administration. A typical preparation will contain from about 5%to about 95% active compound (w/w). Alternatively, such preparationscontain from about 20% to about 80% active compound.

Lower or higher doses than those recited above may be required. Specificdosage and treatment regimens for any particular patient will dependupon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained. Patients may,however, require intermittent treatment on a long-term basis upon anyrecurrence of disease symptoms.

Pharmaceutical compositions of this invention comprise a compound of theformulae described herein or a pharmaceutically acceptable salt thereof;an additional agent including for example, morphine or codeine; and anypharmaceutically acceptable carrier, adjuvant or vehicle. Alternatecompositions of this invention comprise a compound of the formulaedescribed herein or a pharmaceutically acceptable salt thereof; and apharmaceutically acceptable carrier, adjuvant or vehicle. Thecompositions delineated herein include the compounds of the formulaedelineated herein, as well as additional therapeutic agents if present,in amounts effective for achieving a modulation of disease or diseasesymptoms, including BCL-2 family member mediated disorders or symptomsthereof.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, may also beadvantageously used to enhance delivery of compounds of the formulaedescribed herein.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir, preferably by oraladministration or administration by injection. The pharmaceuticalcompositions of this invention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, or carboxymethyl cellulose or similar dispersing agentswhich are commonly used in the formulation of pharmaceuticallyacceptable dosage forms such as emulsions and or suspensions. Othercommonly used surfactants such as Tweens or Spans and/or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms may also be used for the purposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase is combined with emulsifying and/or suspending agents.If desired, certain sweetening and/or flavoring and/or coloring agentsmay be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

When the compositions of this invention comprise a combination of acompound of the formulae described herein and one or more additionaltherapeutic or prophylactic agents, both the compound and the additionalagent should be present at dosage levels of between about 1 to 100%, andmore preferably between about 5 to 95% of the dosage normallyadministered in a monotherapy regimen. The additional agents may beadministered separately, as part of a multiple dose regimen, from thecompounds of this invention. Alternatively, those agents may be part ofa single dosage form, mixed together with the compounds of thisinvention in a single composition.

Screening Assays

The invention provides methods (also referred to herein as “screeningassays”) for identifying polypeptides which modulate the activity of oneor more BCL-2 family proteins or which bind to one or more BCL-2 familyproteins (e.g., a polypeptide having at least one BH homology domain).

The binding affinity of polypeptides described herein can be determinedusing, for example, a titration binding assay. A BCL-2 familypolypeptide or polypeptide comprising a BH domain (e.g., BID, BAK, BAX,etc.) can be exposed to varying concentrations of a candidate compound(i.e., polypeptide) (e.g., 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1mM, and 10 mM) in the presence of a substrate such as a fluorescentlylabeled BH3 containing polypeptide or a fragment thereof (e.g., BID,BAD, BAK, BAX, etc.). The effect of each concentration of candidatecompound is then analyzed to determine the effect of the candidatecompound on BCL-2 family binding activity at varying concentrations,which can be used to calculate the K_(i) of the candidate compound. Thecandidate compound can modulate BCL-2 type activity in a competitive ornon-competitive manner. Direct binding assays can also be performedbetween BCL-2 family proteins and fluorescently labeled candidatecompounds to determine the K_(d) for the binding interaction. Candidatecompounds could also be screened for biological activity in vitro, forexample, by measuring their dose-responsive efficacy in triggeringcytochrome c from purified mitochondria. Cell permeability screeningassays are also envisioned, in which fluorescently labeled candidatecompounds are applied to intact cells, which are then assayed forcellular fluorescence by microscopy or high-throughput cellularfluorescence detection.

The assays described herein can be performed with individual candidatecompounds or can be performed with a plurality of candidate compounds.Where the assays are performed with a plurality of candidate compounds,the assays can be performed using mixtures of candidate compounds or canbe run in parallel reactions with each reaction having a singlecandidate compound. The test compounds or agents can be obtained usingany of the numerous approaches in combinatorial library methods known inthe art.

In one embodiment, an assay is a cell-based assay in which a cell thatexpresses a BCL-2 family protein or biologically active portion thereofis contacted with a candidate polypeptide, and the ability of the testcompound to modulate BCL-2 type activity is determined (e.g., in someinstances increase in apoptosis and in other instances decreaseapoptosis, via intrinsic or extrinsic cell death pathways). Determiningthe ability of the test compound to modulate BCL-2 type activity withincells can be accomplished by monitoring, for example, release ofcytochrome c from the mitochondria or other relevant physiologic readout(e.g., annexin V staining, MTT assay, caspase activity assay, TUNELassay).

In one embodiment, an assay is a biochemical assay, whereby crosslinkedpolypeptides can be linked to affinity resin in order to purify oridentify new or known interactive partners in the apoptotic pathway.

All references cited herein, whether in print, electronic, computerreadable storage media or other form, are expressly incorporated byreference in their entirety, including but not limited to, abstracts,articles, journals, publications, texts, treatises, internet web sites,databases, patents, and patent publications.

Other Applications

Biologically relevant applications for the peptides described herein arenumerous and readily apparent, as indicated by the following cellcompartment-based examples:

-   (1) Cell surface—Natural peptides representing key helical regions    of the HIV-1 protein gp41 (eg. C-peptide, T-20 peptide) have been    shown to prevent viral fusion, and therefore, HIV infectivity.    Helical peptides participate in fusion mechanisms essential to many    virus-host cell infection paradigms (eg. Dengue, Hepatitis C,    Influenza), and therefore, hydrocarbon-stapled analogues of these    critical helical regions may function as effective antibiotics by    inhibiting viral fusion. In general, ligands that interact with cell    surface receptors using helical interfaces to activate or inhibit    signaling pathways, represent additional applications for the    polypeptides described herein.-   (2) Intramembrane—Receptor dimerization and oligomerization are    cardinal features of ligand-induced receptor activation and    signaling. Transmembrane helical domains widely participate in such    essential oligomerization reactions (eg. Epidermal Growth Factor    Receptor [EGFR] family), and specific peptide sequences have been    defined that facilitate these tight intramembrane helical    associations. Aberrant activation of such receptors through    oligomerization are implicated in disease pathogenesis (eg. erbB and    cancer). Therefore, in the appropriate setting, activation or    inhibition of transmembrane inter-helical interactions would have    therapeutic benefit.-   (3) Cytosolic—Cytosolic targets include soluble protein targets and    those associated with specific intracytosolic organelles, including    the mitochondria, endoplasmic reticulum, Golgi network, lysosome,    and peroxisome. Within the field of apoptosis, there are multiple    cytosolic and mitochondrial apoptotic protein targets for    hydrocarbon-stapled BCL-2 family domains. Within the BH3-only    subgroup of pro-apoptotic proteins, two major subsets of BH3 domains    have been identified: (1) BID-like BH3s (e.g., BIM) which are    apoptosis “activators,” inducing BAK oligomerization and cytochrome    c release at the mitochondrion and (2) BAD-like BH3s which are    apoptosis “sensitizers” that selectively target anti-apoptotic    multidomain proteins, enabling subliminal levels of activating    domains to be maximally effective. In addition to distinct binding    of BH3-only proteins to pro- vs. anti-apoptotic multidomain family    members, BH3 domains display differential binding among    anti-apoptotic proteins. For example, it has been demonstrated that    BAD preferentially binds to the anti-apoptotic BCL-2, whereas BIM    targets the anti-apoptotic MCL-1. Identifying and exploring these    selective interactions are critically important because different    BCL-2 family members are implicated in different types of cancer.    For example, BCL-2 overexpression is responsible for the development    of follicular lymphoma and chemotherapy resistance in general,    whereas MCL-1 is believed to play an important role in the    pathogenesis of multiple myeloma. The ability to transform the many    BH3 domains into structurally stable and cell permeable reagents    would provide an important opportunity to explore and differentially    manipulate apoptotic pathways in cancer cells. Targeting further    helix-dependent interactions in the cytosol or at cytosolic    organelles is envisioned.-   (4) Nuclear—Nuclear transcription factors and their modulatory    proteins drive a host of physiologic processes based upon peptide    helical interactions with nuclear proteins and nucleic acids. The    feasibility of generating hydrocarbon-stapled peptides to engage in    nuclear interactions has recently been demonstrated by our synthesis    of a panel of hydrocarbon-stapled p53 peptides, which interact with    MDM2 at picomolar affinities. In addition to modulating    protein-protein interactions within the nucleus, protein-nucleic    acid interactions are also apparent targets. Multiple transcription    factor families, such as homeodomain, basic helix-loop-helix,    nuclear receptor, and zinc finger-containing proteins, directly    interact with DNA via their peptide helices to activate or inhibit    gene transcription. As an example, homeodomain proteins are a family    of essential transcription factors that regulate genetic programs of    growth and differentiation in all multicellular organisms. These    proteins share a conserved DNA-binding motif, called the    homeodomain, which contains a 60 amino acid long peptide that forms    three a-helices, the third of which makes direct contact with the    major groove of DNA. Like the BH3 domain of apoptotic proteins, the    homeodomain is a critical effector motif with sufficient variation    among homologs to facilitate differential binding specificities and    physiologic activities. Protein-DNA interactions can be complex and    extensive, and thereby present a challenge to small molecule    development for the purpose of studying and selectively modulating    transcriptional events. In higher organisms, homeodomain proteins    are highly expressed during development, specifying the body plan    and dictating tissue differentiation. Overexpression of specific    homeoproteins (eg. CDX4) can activate tissue-specific    differentiation programs resulting in, for example, blood formation    from mouse embryonic stem cells. Deregulation of homeotic gene    expression, such as aberrant upregulation of homeodomain proteins    typically expressed in undifferentiated cells or inappropriate    downregulation of such proteins normally expressed in differentiated    cells, can contribute to the development and maintenance of cancer.    For example, in pediatric alveolar rhabdomyosarcoma, fusion of the    PAX3 or PAX 7 DNA binding domain to the transactivating domain of    forkhead has been implicated in cellular transformation;    translocations involving the DNA-binding domains of several HOX    genes have been linked to the pathogenesis of leukemia. Thus, the    ability to chemically-stabilize transcription factor helices, such    as homeodomain peptides, for cellular delivery has the potential to    yield a chemical toolbox for the investigation and modulation of    diverse transcription programs responsible for a multitude of    biological process in health and disease.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

The invention claimed is:
 1. A cell-penetrable cross-linked polypeptideof Formula (I):

wherein: each R₁ and R₂ are independently H, C₁-C₂₀ alkyl, C₂-C₂₀alkenyl, C₂-C₂₀ alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, orheterocyclylalkyl; wherein at least either R₁ or R₂ is C₁-C₂₀ alkyl,C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, arylalkyl, cycloalkylalkyl,heteroarylalkyl, or heterocyclylalkyl; R₃ is a covalent crosslinkspanning from one to two turns of an alpha helix; x is an integerselected from 2 to 6; each y is independently an integer from 0-100; zis an integer from 1-10; and each Xaa is independently an amino acid,wherein the polypeptide has a substantially alpha helical secondarystructure in aqueous solution and penetrates a cell membrane.
 2. Thecell-penetrable cross-linked polypeptide of claim 1, wherein thepolypeptide is transported or actively transported through the cellmembrane, as determined in a fluorescent assay.
 3. The cell-penetrablecross-linked polypeptide of claim 2, wherein the fluorescent assaycomprises fluorescently-labeling the cell-penetrable cross-linkedpolypeptide, applying the fluorescently-labeled cell-penetrablecross-linked polypeptide to an intact cell, and assaying the cell forcellular fluorescence by microscopy or high-throughput cellularfluorescence detection.
 4. The cell-penetrable cross-linked polypeptideof claim 1, wherein the cell-penetrable cross-linked polypeptide hasenhanced cell penetrability relative to a corresponding uncrosslinkedpolypeptide.
 5. The cell-penetrable cross-linked polypeptide of claim 1,wherein the cell-penetrable cross-linked polypeptide displays enhancedalpha-helicity compared to a corresponding uncrosslinked polypeptide. 6.The cell-penetrable cross-linked polypeptide of claim 1, wherein x is 2,3, or
 6. 7. The cell-penetrable cross-linked polypeptide of claim 1,wherein z is
 1. 8. The cell-penetrable cross-linked polypeptide of claim1, wherein each y is independently an integer between 3 and
 15. 9. Thecell-penetrable cross-linked polypeptide of claim 1, wherein R₁ and R₂are each independently C₁-C₆ alkyl.
 10. The cell-penetrable cross-linkedpolypeptide of claim 1, wherein R₁ or R₂ is H.
 11. The cell-penetrablecross-linked polypeptide of claim 1, wherein at least one of R₁ and R₂is methyl.
 12. The cell-penetrable cross-linked polypeptide of claim 1,wherein: R₃ is alkyl, alkenyl, alkynyl, [R₄—K—R₄]_(n), or a naturallyoccurring amino acid side chain, wherein R₃ is substituted with 0-6 R₅;R₄ is alkyl, alkenyl, or alkynyl; R₅ is halo, alkyl, OR₆, N(R₆)₂, SR₆,SOR₆, SO₂R₆, CO₂R₆, R₆, an epoxide, a fluorescent moiety, or aradioisotope; K is O, S, SO, SO₂, CO, CO₂, CONR₆, or

R₆ is H, alkyl, or a therapeutic agent; and n is an integer from 1-4.13. The cell-penetrable cross-linked polypeptide of claim 12, wherein R₃is alkyl or alkenyl.
 14. A method of promoting apoptosis of a neoplasticcell in a subject in need thereof, the method comprising administeringto the subject a therapeutically effective amount of a helicalcross-linked polypeptide derived from a protein involved in a cellularapoptotic pathway, wherein the cross-linked polypeptide is of Formula(I):

wherein: each R₁ and R₂ are independently H, C₁-C₂₀ alkyl, C₂-C₂₀alkenyl, C₂-C₂₀ alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, orheterocyclylalkyl; wherein at least either R₁ or R₂ is C₁-C₂₀ alkyl,C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, arylalkyl, cycloalkylalkyl,heteroarylalkyl, or heterocyclylalkyl; R₃ is a covalent crosslinkspanning from one to two turns of an alpha helix; x is an integerselected from 2 to 6; each y is independently an integer from 0-100; zis an integer from 1-10; and each Xaa is independently an amino acid.15. The method of claim 14, wherein the cross-linked polypeptidedisplays enhanced alpha-helicity compared to a correspondinguncrosslinked polypeptide.
 16. The method of claim 14, wherein thecross-linked polypeptide displays enhanced cell permeability compared toa corresponding uncrosslinked polypeptide.
 17. The method of claim 14,wherein the cross-linked polypeptide comprises an alpha-helical domainof a BCL-2 family member.
 18. The method of claim 14, wherein thecross-linked polypeptide comprises an alpha-helical BH3 domain of aBCL-2 family member.
 19. The method of claim 14, wherein x is 2, 3, or6.
 20. The method of claim 14, wherein z is
 1. 21. The method of claim14, wherein each y is independently an integer between 3 and
 15. 22. Themethod of claim 14, wherein R₁ and R₂ are each independently C₁-C₆alkyl.
 23. The method of claim 14, wherein at least one of R₁ and R₂ ismethyl.
 24. The method of claim 14, wherein R₁ or R₂ is H.
 25. Themethod of claim 14, wherein: R₃ is alkyl, alkenyl, alkynyl,[R₄—K—R₄]_(n), or a naturally occurring amino acid side chain, whereinR₃ is substituted with 0-6 R₅; each R₄ is independently alkyl, alkenyl,or alkynyl; R₅ is halo, alkyl, OR₆, N(R₆)₂, SR₆, SOR₆, SO₂R₆, CO₂R₆, R₆,an epoxide, a fluorescent moiety, or a radioisotope; K is O, S, SO, SO₂,CO, CO₂, CONR₆, or

R₆ is H, alkyl, or a therapeutic agent; and n is an integer from 1-4.26. The method of claim 25, wherein R₃ is alkyl or alkenyl.
 27. Apharmaceutical composition comprising the cell-penetrable cross-linkedpolypeptide of claim 1, and a pharmaceutically acceptable excipient. 28.The cell-penetrable cross-linked polypeptide of claim 1, wherein each R₁and R₂ are independently C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl,arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl. 29.The cell-penetrable cross-linked polypeptide of claim 1, wherein each R₁are independently C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl,arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl. 30.The cell-penetrable cross-linked polypeptide of claim 1, wherein thecross-linked polypeptide displays increased permeability compared to apolypeptide that is not cross-linked.
 31. The cell-penetrablecross-linked polypeptide of claim 1, wherein each R₂ are independentlyC₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, arylalkyl,cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl.
 32. The methodof claim 14, wherein the neoplastic cell is a fibrosarcoma, myosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyo sarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreaticcancer, ovarian cancer, prostate cancer, uterine cancer, cancer of thehead and neck, skin cancer, brain cancer, squamous cell carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcancer, testicular cancer, small cell lung carcinoma, non-small celllung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposisarcoma cell.